The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain
For nearly six decades , we have known that lipoprotein lipase ( LPL ) , a triglyceride hydrolase secreted by myocytes and adipocytes , is essential for the lipolytic processing of triglyceride-rich lipoproteins ( TRLs ) in the bloodstream ( Korn , 1955; Young and Zechner , 2013 ) . LPL-mediated processing of TRLs occurs along the capillary lumen , and LPL is readily released from the surface of capillaries with polyanionic compounds such as heparin . LPL contains an N-terminal domain ( NTD ) for catalysis and a C-terminal domain ( CTD ) that is essential for lipid binding . LPL is active as a head-to-tail homodimer , and several lines of evidence suggest that the CTD of one LPL monomer delivers triglyceride substrates to the NTD of the partner monomer ( Kobayashi et al . , 2002 ) . In the presence of certain LPL mutations ( e . g . , mutations in the catalytic triad or mutations that prevent LPL dimerization ) , the processing of TRLs is markedly impaired , leading to severe hypertriglyceridemia ( familial chylomicronemia ) ( Brahm and Hegele , 2015 ) . For decades , the mechanism by which LPL traversed endothelial cells to reach the luminal surface of capillaries—as well as LPL’s binding site within capillaries—were enigmas ( Brown et al . , 2015 ) . These uncertainties have been addressed by the discovery of a novel LPL binding protein of capillary endothelial cells , glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 ( GPIHBP1 ) . GPIHBP1 binds LPL within the interstitial spaces and transports LPL across endothelial cells to its site of action in the capillary lumen ( Beigneux et al . , 2007; Davies et al . , 2010 ) . In the absence of GPIHBP1 , LPL does not reach the capillary lumen , and there is no margination of TRLs along the surface of capillaries ( Goulbourne et al . , 2014 ) . GPIHBP1 is a member of the LU ( Ly6/uPAR ) protein domain family , which includes CD59 , SLURP1 , C4 . 4A , TGF-β receptors , and uPAR . The hallmark of this protein family is a three-finger–fold with a defined disulfide pairing motif involving eight highly conserved cysteines ( Ploug , 2003 ) . Members of the LU domain family are widespread in the animal kingdom and occur as secreted proteins ( e . g . SLURP1 ) , integral membrane proteins ( TGFβ receptors ) , and glycolipid-anchored proteins ( e . g . CD59 , GPIHBP1 , C4 . 4A , uPAR ) . During evolution , the LU domain has evolved to serve many different purposes in mammals: GPIHBP1 transports LPL; CD59 inhibits complement activation; TGFβ receptors are involved in cytokine signaling; TEX101 regulates fertility; and uPAR focuses plasminogen activation on cell surfaces . Although detailed structure–function relationships have been elucidated for a few mammalian LU domain proteins ( e . g . TGFβ receptors , CD59 , uPAR ) , progress in understanding the biochemistry and biophysics of protein interactions for the majority of LU domain proteins , including GPIHBP1 , has lagged behind , largely because of the need for highly purified recombinant protein preparations . In our opinion , GPIHBP1 represents one of the more intriguing mammalian LU domain proteins from both a functional and a structural point of view . First , GPIHBP1 function is tightly linked to human disease as a variety of loss-of-function mutations in GPIHBP1 have been encountered in humans with familial chylomicronemia ( Beigneux et al . , 2009; Buonuomo et al . , 2015; Olivecrona et al . , 2010; Plengpanich et al . , 2014; Rios et al . , 2012; Surendran et al . , 2012 ) . The majority of these disease-causing mutations impair the folding of the LU domain , leading to multimerized and dysfunctional GPIHBP1 molecules on the cell surface ( Beigneux et al . , 2015 ) . In a comprehensive screening of GPIHBP1 mutants , one mutant ( GPIHBP1W89S ) was found to have markedly impaired LPL binding despite being monomeric , sugge sting that it retained the native folding of its LU domain ( Beigneux et al . , 2011; Beigneux et al . , 2015 ) . Interestingly , several mutations in the CTD of LPL ( e . g . C418Y , first identified in a patient with chylomicronemia ) have no effect on catalysis but abolish the ability of LPL to bind to GPIHBP1 ( and thus prevent LPL transport across endothelial cells ) ( Gin et al . , 2012; Henderson et al . , 1996 ) . Amino acid numbering throughout this article refers to the first residue in the mature protein . Second , GPIHBP1 is unique from a structural perspective . Among the entire LU protein family , only very few proteins have N-terminal extensions ( e . g . GPIHBP1 and the spermatid marker SP-10 ) . In the case of GPIHBP1 , the N-terminal extension is highly enriched in acidic residues , with 21 of 26 consecutive amino acids in human GPIHBP1 being aspartic acid or glutamic acid . Early studies in cell culture suggested that GPIHBP1’s acidic domain is important for GPIHBP1•LPL interactions ( Gin et al . , 2008 ) , but a detailed study of the functional impact of the acidic domain on LPL function has been lacking . One of the impediments to progress is that a large fraction of the GPIHBP1 produced by transfected mammalian cells is misfolded and in the form of multimers ( Beigneux et al . , 2015 ) . In the current study , we used highly purified proteins and a combination of hydrogen–deuterium exchange mass spectrometry ( HDX-MS ) , surface plasmon resonance ( SPR ) , zero-length cross-linking , and LPL activity assays to elucidate LPL•GPIHBP1 interactions both kinetically and dynamically . Our studies show: ( 1 ) that the kinetics of LPL•GPIHBP1 interactions are controlled by both the LU domain and the acidic domain of GPIHBP1; and ( 2 ) that the acidic domain is intrinsically disordered and is responsible for stabilizing the catalytic activity of LPL by inhibiting the inherent instability and subsequent unfolding of LPL’s catalytic domain .
A secreted version of human GPIHBP11–131 was produced in Drosophila S2 cells as a fusion protein with uPAR domain III ( Figure 1—figure supplement 1 ) . However , this protein proved to be prone to an internal cleavage after Arg38 during enterokinase-mediated removal of the uPAR tag . This unexpected cleavage event markedly reduced the yields of purified GPIHBP11–131 . Alignments of multiple primate GPIHBP1 sequences revealed that Arg38 is not conserved during evolution . In GPIHBP1 from Nomascus leucogenys , which is 94% identical to human GPIHBP1 , the residue corresponding to Arg38 is Gly38 . Based on these homology considerations , we therefore expressed and purified a modified protein in which Arg38 was replaced with Gly; this construct yielded high levels of pure GPIHBP11–131/R38G along with moderate amounts of a truncated GPIHBP134–131/R38G . That truncated protein was the result of an additional cleavage after Arg33 . Both GPIHBP11–131 and GPIHBP134–131 ( lacking the acidic domain ) were purified to homogeneity by cation-exchange chromatography ( Figure 1—figure supplement 2 ) . Importantly , both proteins were monomeric with no traces of aggregation , as judged by analytical size-exclusion chromatography ( Figure 1C ) . This homogeneity is a noteworthy achievement because earlier studies had shown that GPIHBP1 is highly susceptible to multimerization ( Beigneux et al . , 2015 ) . The anomalous partitioning of GPIHBP11–131 during size-exclusion chromatography is most likely a consequence of a large Stokes radius caused by the presence of an intrinsically disordered N-terminal peptide ( see next section ) . Consistent with this assumption , GPIHBP134–131 , which lacks the acidic domain , eluted with the expected hydrodynamic volume for a globular protein ( Figure 1C ) . 10 . 7554/eLife . 12095 . 003Figure 1 . Model of human GPIHBP1 . Panel A shows a cartoon representation for glycolipid-anchored human GPIHBP1 . Predicted β-sheets are shown in cyan; acidic amino acid residues in the N-terminal domain are highlighted by red sticks; and the consensus disulfide bonds are shown in yellow . Panel B shows a “disorder prediction” for human GPIHBP1 sequence based on the IUPred algorithm . Locations of the six strands of the three-fingered–fold of the LU domain are highlighted by boxes ( A–F ) ; strands predicted to form β-sheets are colored cyan . Panel C documents the homogeneity and monomer status of purified GPIHBP11–131/R38G ( #A ) and GPIHBP134–131/R38G ( #B ) by analytical size-exclusion chromatography with a Superdex HR75 column operated with 20 mM NaH2PO4 and 150 mM NaCl ( pH 7 . 2 ) and SDS-PAGE ( inset ) . Elution positions of the calibration standards are indicated by blue arrows: ovalbumin ( 43 kDa ) , carbonic anhydrase ( 29 kDa ) , ribonuclease ( 13 kDa ) , and aprotenin ( 6 . 5 kDa ) . Panel D provides a heat map representation of the relative deuterium uptakes ( relative to a fully exchanged control ) in peptic peptides from free and LPL-occupied GPIHBP11–131 as assessed by HDX-MS . Deuterium uptake was measured after 10 , 100 , and 1000 s incubations in D2O , and relative deuterium uptake is assigned according to the color code ( ranging from blue , no deuterium uptake , to red , full deuterium uptake ) . A cartoon representation of the differential deuterium uptake for the LU domain between free and LPL-occupied GPIHBP1 after a 10-s exchange is shown in panel E . Shown as sticks are the positions of Thr85 , Ser87 , and Trp89 in β-strand D of GPIHBP1 . Raw deuterium uptake values for peptides 1–21 and 83–109 are shown for free and LPL-occupied GPIHBP1 ( blue and red solid lines , respectively ) . The dashed line represents a “full deuteration” control . GPI , glycosylphosphatidylinositol; MS , hydrogen–deuterium exchange mass spectrometry; LU , Ly6/uPAR; SDS-PAGE , sodium dodecyl sulfate polyacrylamide gel electrophoresisDOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 00310 . 7554/eLife . 12095 . 004Figure 1—figure supplement 1 . Constructs encoding soluble DIII-ent-hGPIHBP11–131/R38G and DIII-ent-hLPL313–438 . Schematic representation of the vectors used for Drosophila S2 cell expression of a soluble , secreted human GPIHBP11–131 ( panel A ) and the CTD313–448 of human LPL ( panel B ) . Both constructs use the third LU domain from human uPAR ( DIII192–283 ) as an N-terminal purification tag as described ( Gårdsvoll et al . , 2013 ) . The enterokinase cleavage site used to excise the recombinant protein ( and remove the uPAR tag ) is highlighted in black . pMT and BiP represent the Drosophila metallothionein promoter and secretion signals , respectively . To optimize enterokinase-mediated excision of GPIHBP1 , two mutations ( DIIIR281G and GPIHBP1R38G ) were introduced to silence two undesired cleavage sites for enterokinase . In the GPIHBP1 construct , the N-terminal signal peptide and the C-terminal hydrophobic peptide required for glycolipid anchoring have been omitted . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 00410 . 7554/eLife . 12095 . 005Figure 1—figure supplement 2 . Purification of recombinant soluble human GPIHBP11–131/R38G and GPIHBP134–131/R38G . Panel A shows a cation-exchange chromatography elution profile for 30 mg of purified DIII-ent-GPIHBP11–131/R38G after enterokinase cleavage . We used a 5 ml HiTrap SP FF column and a 35-ml linear gradient from 0 to 1 . 0 M NaCl in 50 mM CH3COOH , pH 4 . 5 . The peaks correspond to: intact GPIHBP11–131/R38G ( #A ) , GPIHBP134–131/R38G ( #B ) , uncleaved fusion protein ( #C ) and the excised uPAR DIII tag ( #D ) , as judged by SDS-PAGE of reduced and alkylated samples ( panel B ) and mass spectrometry ( not shown ) . SDS-PAGE , sodium dodecyl sulfate polyacrylamide gel electrophoresis . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 00510 . 7554/eLife . 12095 . 006Figure 1—figure supplement 3 . Comparison of the dynamics in GPIHBP134–131 and GPIHBP11–131 in HDX-MS experiments . Differential deuterium uptake between intact GPIHBP11–131 and GPIHBP134–131 ( a truncated GPIHBP1 lacking the N-terminal acidic domain ) measured by HDX-MS at 25°C is shown as a butterfly plot . Deuterium uptake was traced for 10- ( orange ) , 100- ( black ) , and 1000-sec ( blue ) . The shaded gray area corresponds to the largest standard deviation of the data sets recorded for each peptide . Data points represent the mean of triplicate measurements . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 00610 . 7554/eLife . 12095 . 007Figure 1—figure supplement 4 . Peptide list for pepsin-treated GPIHBP1 . Peptides recovered after HDX and pepsin cleavage of recombinant human GPIHBP1 are shown as blue bars . HDX , hydrogen–deuterium exchange . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 007 Homology considerations identify GPIHBP1 as a glycolipid-anchored protein with a single prototypical LU domain ( Figure 1A ) . Unlike other members of this protein family , GPIHBP1 contains an N-terminal domain with 21 acidic amino acids ( Glu , Asp ) . We propose that the first 30–35 N-terminal residues in GPIHBP1 have a very high propensity for being an intrinsically disordered region ( Figure 1B ) . The disordered nature of the acidic domain is supported by: ( 1 ) the atypically large hydrodynamic volume of GPIHBP11–131 compared with GPIHBP134–131 ( Figure 1C ) ; ( 2 ) its susceptibility to limited proteolysis after Arg33 or Arg38; and ( 3 ) by the highly dynamic nature of the acidic domain as judged by extremely rapid hydrogen–deuterium exchange rates . In hydrogen–deuterium exchange/mass spectrometry ( HDX-MS ) studies , we observed 100% deuterium uptake in this domain even after the shortest exposure time ( 10 s ) ( Figure 1D and 1E ) , which is consistent with the predicted deuterium uptake for a disordered GPIHBP11–33 peptide ( 87% after 1 s and 100% after 10 s ) . In the same experiment , HDX profiles of peptides within the LU domain followed the secondary structure prediction . The deuterium uptake in the isolated LU domain in GPIHBP134–131 is indistinguishable from that of full-length GPIHBP11–131 ( Figure 1—figure supplement 3 ) , which implies that the acidic N-terminal region has little or no effect on the structure of GPIHBP1’s LU domain . To identify protein–protein binding interfaces and/or uncover conformational changes associated with GPIHBP1•LPL binding , we determined the hydrogen–deuterium exchange profiles for GPIHBP1 , LPL , and GPIHBP1•LPL complexes . The GPIHBP1•LPL complexes were formed by incubating 5 µM GPIHBP1 with 5 µM LPL homodimers ( LPL2 ) for 15 min in 10 mM Na2HPO4 , 150 mM NaCl ( pH 7 . 4 ) at 25°C before monitoring solvent exchange after dilution into D2O for 10 , 100 , or 1000 s . We recovered 22 peptides from GPIHBP1 and 92 peptides from LPL after on-line pepsin digestion of quenched and tris ( 2-carboxyethyl ) phosphine ( TCEP ) -reduced proteins at pH 2 . 5 , which correspond to 100% and 87% sequence coverage , respectively ( Figure 1–figure supplement 4 and Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 12095 . 008Figure 2 . Mapping GPIHBP1 interaction sites on LPL . Differential deuterium uptake values for free LPL and LPL occupied with intact GPIHBP11–131 ( panel A ) or the acidic domain peptide GPIHBP11–33 ( panel B ) are shown as butterfly representations . The butterfly plot in panel C compares LPL occupied with GPIHBP11–131 or GPIHBP11–33 . Due to inherent instability of LPL homodimers , the deuterium uptake was only examined at 10- ( orange ) and 100-s ( black ) incubations . The data points represent the mean of triplicate measurements , and the shaded gray area corresponds to the largest standard deviation in the data sets recorded for each peptide . A total of 92 peptides were recovered from LPL , and they are numbered consecutively from the N-terminus . The sequences of the individual peptides are found in Figure 2—figure supplement 1B . The transparent red and cyan colors on the left in panel A localize peptides to either the NTD or the CTD domains of LPL , respectively . Panel D shows a cartoon representation of human LPL . Two regions having the most pronounced changes in deuterium uptake with GPIHBP1 binding are highlighted in green ( residues 279–293 ) and blue ( residues 402–419 ) ; basic residues are shown as sticks . CTD , C-terminal domain; LPL , lipoprotein lipase; NTD , N-terminal domain . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 00810 . 7554/eLife . 12095 . 009Figure 2—figure supplement 1 . Peptide list for pepsin-treated bovine LPL . Panel A shows peptides recovered in the HDX-MS studies . Peptides recovered from bovine LPL after pepsin cleavage are shown as blue bars . The CTD is highlighted by the transparent light green box; N-linked glycosylation sites are shown ( CHO ) ; residues forming the catalytic triad are indicated by blue asterisks; heparin-binding sequences are shown by green asterisks . Panel B shows the inventory of 92 peptides recovered from LPL . Peptides designated with an asterisk display a bimodality of their isotopic envelopes after a 45-min incubation in H2O-buffers followed by 10 and 100 s incubations in D2O buffers ( as illustrated for the peptic peptide 131–165 in Figure 6A ) . CHO , carbohydrate; CTD , C-terminal domain; HDX-MS , hydrogen–deuterium exchange mass spectrometry; LPL , lipoprotein lipase . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 00910 . 7554/eLife . 12095 . 010Table 1 . Kinetic rate constants for GPIHBP1 interactions with CTD and LPL homodimers . aDOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 010kon ( 105 M–1s–1 ) koff ( s–1 ) KD ( µM ) nCaptureGPIHBP11–131/R38G10 . 4 ± 1 . 90 . 12 ± 0 . 040 . 12 ± 0 . 0411CTDGPIHBP11–13111 . 2 ± 4 . 00 . 22 ± 0 . 070 . 29 ± 0 . 1310CTDGPIHBP134–131/R38G1 . 0 ± 0 . 60 . 10 ± 0 . 011 . 41 ± 0 . 764CTDGPIHBP11–131/W89Snbdnbdnbd2CTDGPIHBP11–131/R38G N-glyb10 . 60 . 160 . 152CTDGPIHBP11–131/R38G9 . 9 ± 5 . 00 . 023 ± 0 . 0060 . 025 ± 0 . 0078LPLGPIHBP11–1316 . 5 ± 4 . 40 . 020 ± 0 . 0070 . 038 ± 0 . 0298LPLGPIHBP134–131/R38G1 . 9 ± 0 . 40 . 019 ± 0 . 0050 . 091 ± 0 . 0124LPLGPIHBP11–131/R38Gnbdnbdnbd1CTDC418YGPIHBP134–131/R38Gnbdnbdnbd1CTDC418YaThe kinetic rate constants ( kon and koff ) were derived by global fitting of sensorgrams obtained by either single-cycle kinetics ( LPL ) or a mixture of single- and multi-cycle kinetics ( the CTD of LPL ) ; the dissociation equilibrium constant KD was calculated as kon/koff . bIntact GPIHBP11–131/R38G ( 2 nmols ) was incubated overnight at 37°C with 2 U N-glycanase under native conditions . The deglycosylated protein remained monomeric , as judged by analytical size-exclusion chromatography ( as performed in Figure 1C ) . n , number of separate determinations , each involving a complete set of analyte concentrations analyzed on different days and/or different CM4 chips; nbd , no specific binding detected; CTD , C-terminal domain; LPL , lipoprotein lipase . 10 . 7554/eLife . 12095 . 011Figure 3 . Real-time binding kinetics for the interaction between GPIHBP1 and the CTD of human LPL . Panel A shows repeat binding of 100 nM of recombinant CTD313–448 from human LPL to immobilized mAb 5D2 followed by multi-cycle injections of twofold dilutions of GPIHBP11–131 ( black lines ) . The green line represents a buffer control; the blue line represents a repeat injection of 250 nM GPIHBP1 at the end of the experiment to demonstrate reproducibility . Panel B shows equilibrium binding isotherms for the interactions between the immobilized CTD of LPL and GPIHBP11–131 ( blue ) , GPIHBP134–131 ( red ) , and GPIHBP11–131/W89S ( green ) with the SPR signal at 380 s in panel A as equilibrium binding level . Panel C shows a kinetic evaluation of the double-referenced SPR data for GPIHBP11–131 with a global fit to a 1:1 binding model ( fits shown in red ) . Note there is a slight decay of the binding signal at equilibrium due to a weak ligand-induced dissociation of the CTD from mAb 5D2; this effect translates into dissociation below baseline for the higher concentration of GPIHBP11–131 . These effects were not observed for GPIHBP134–131 . CTD , C-terminal domain; LPL , lipoprotein lipase; SPR , surface plasmon resonance . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 01110 . 7554/eLife . 12095 . 012Figure 4 . Kinetic assessment of the GPIHBP1•LPL interaction by single-cycle titration SPR . The basic principles in the single-cycle kinetic titration are illustrated in panel A . Initially , LPL is captured in a noncovalent fashion on the CM4 sensor surface after a 150 s injection of 200 nM of purified LPL across the flow cell containing immobilized mAb 5D2 . After a 600 s stabilization period , a series of five 90 s pulses with increasing analyte concentration are injected without intervening regeneration . The following concentrations of either mAb 4-1a ( A ) , mAb 5D2 ( A ) , GPIHBP11–131 ( B ) , or GPIHBP134–131 ( C ) were analyzed: 12 . 5 , 25 , 50 , 100 , and 200 nM . Panels B and C shows the buffer referenced sensorgrams recorded for GPIHBP11–131 and GPIHBP134–131 , respectively . The mathematical fitting to a simple 1:1 binding model are superimposed as red lines and the residuals are shown in green . LPL , lipoprotein lipase; SPR , surface plasmon resonance . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 012 As documented by the heat maps in Figure 1D , the conformation of the LU domain in GPIHBP11–131 was less dynamic when bound to LPL . In particular , we observed markedly reduced deuterium uptake within strands D and E of GPIHBP1 when it formed a complex with LPL ( Figure 1E ) . This shift in the dynamics likely reflects a transition to a state with increased secondary structure and/or a direct engagement in a ligand-binding interface . The fact that the HDX-MS analysis of GPIHBP1 identified β-strand D as a candidate binding interface for LPL was not entirely unexpected given that a previous mutagenesis study had shown that individual alanine replacements of Thr85 , Ser87 , or Trp89 in β-strand D impaired LPL binding ( Figure 1E ) ( Beigneux et al . , 2011 ) . Moreover , two missense mutations involving β-strand D have been associated with chylomicronemia in humans ( i . e . Ser87→Pro [Buonuomo et al . , 2015] and Thr88→Arg [Surendran et al . , 2012] ) . Mutating residues within strand E of GPIHBP1 had little impact on LPL binding ( Beigneux et al . , 2011 ) . The reduced deuterium uptake that we recorded for strand E in the presence of LPL probably relates to a higher propensity for β-sheet formation in the setting of the GPIHBP1•LPL complex . Strand E is one of the most versatile structures in LU proteins; it can adopt a random coil ( snake venom α-neurotoxins ) , a β-strand ( uPAR domains I and II ) , or an α-helix ( CD59 , Prod1 , and uPAR domain III ) ( Kriegbaum et al . , 2011 ) . Interestingly , the deuterium uptake in the intrinsically disordered N-terminal acidic domain did not change with LPL binding . That finding suggests that the acidic domain either does not participate in the binding interaction or more likely is involved in very transient binding events with LPL ( interactions that do not lead to the formation of stable hydrogen bonds ) . From the same set of experiments , we also extracted data concerning the effects on LPL by comparing the differential deuterium uptake between unoccupied LPL and LPL in complex with either intact GPIHBP11–131 ( Figure 2A ) or its N-terminal acidic domain peptide GPIHBP11–33 ( Figure 2B ) . To minimize possible confounding effects from the inherent instability of unoccupied LPL , we only included deuterium uptake values for 10 and 100 s for the three different states of LPL . Comparing these states , we identified residues 402–419 in the CTD of LPL as the most likely interaction site for the LU domain of GPIHBP1 ( shown in blue in the butterfly plot in Figure 2A ) . This assignment is based on the fact that GPIHBP11–131 , but not GPIHBP11–33 , attenuates the deuterium uptake in this particular region ( Figure 2C ) . Two missense mutations in or close to this segment ( C418Y and E421K ) were identified in patients with chylomicronemia ( Henderson et al . , 1998; Henderson et al . , 1996 ) , and follow-up studies showed that both mutations impaired GPIHBP1 binding ( Voss et al . , 2011 ) . It is possible that additional regions in the CTD of LPL are involved in GPIHBP1 binding but escaped detection in our HDX-MS experiments due to the lower level of sequence coverage for the CTD ( 69% ) ( Figure 2—figure supplement 1 ) . The impact of the acidic domain peptide ( GPIHBP11–33 ) on LPL deuterium uptake was less pronounced than with intact GPIHBP11–131 ( Figure 2A and 2B ) , consistent with a more dynamic interaction ( i . e . a shorter residence time of the peptide on LPL ) . Notwithstanding the more transient interaction , we did nevertheless observe that GPIHBP11–33 reduced deuterium uptake in LPL peptides spanning residues 279–293 ( Figure 2B ) , which is localized in the domain interface between the NTD and CTD of LPL ( Figure 2D ) . It is noteworthy that this region contains one of the heparin-binding sites in LPL enriched in basic residues ( Arg281 , Lys282 , Arg284 ) ( Hata et al . , 1993 ) and therefore represents a potential binding partner for GPIHBP1’s acidic domain . To dissect the individual contributions of the folded LU domain and the intrinsically disordered acidic domain on the binding kinetics with LPL , we developed and optimized a SPR assay to measure this interaction . In brief , we used a BiacoreT200 to measure the binding kinetics between soluble , monomeric GPIHBP1 and either LPL’s CTD or intact LPL that had been captured on a CM4 sensor chip with an immobilized anti-LPL monoclonal antibody , 5D2 . This antibody is well suited to capture and display both LPL and the CTD in a defined orientation because it recognizes both proteins with high affinity ( Chang et al . , 1998 ) . Using this SPR setup , we found that full-length GPIHBP11–131/R38G binds the CTD of LPL with a KD of 0 . 12 ± 0 . 04 µM , whereas GPIHBP134–131/R38G binds with a KD of 1 . 41 ± 0 . 76 µM ( Table 1 ) . The robustness of the assay is apparent by the repeat analyses of 250 nM GPIHBP1 at the end of each multi-cycle segment ( blue curves in Figure 3A and 3C ) . In parallel , we analyzed the ability of soluble GPIHBP1 to bind to a CTD carrying a single amino acid substitution ( C418Y ) . As noted earlier , the C418Y mutation is known to abolish LPL binding to GPIHBP1 ( Henderson et al . , 1996; Voss et al . , 2011 ) . When the LPL CTDC418Y was captured on immobilized mAb 5D2 ( 85 RU ~ 5 fmols/mm2 ) , it lacked the ability to bind GPIHBP11–131 or GPIHBPB134–131 , even at concentrations as high as 1 µM ( Table 1 ) . As an additional control , we tested the ability of GPIHBP11–131/W89S to bind the CTD of LPL . Earlier studies had shown that glycolipid-anchored GPIHBP1W89S on the surface of transfected CHO-K1 cells has little or no capacity to bind LPL ( Beigneux et al . , 2011; Beigneux et al . , 2015; Plengpanich et al . , 2014 ) . Consistent with those studies , the SPR measurements revealed negligible binding of GPIHBP11–131/W89S to the CTD of LPL ( Figure 3B ) . Collectively , these data indicate that the intrinsically disordered acidic domain of GPIHBP1 has little effect on the affinity of the LPL•GPIHBP1 interaction in the absence of a functional LU domain . Kinetic assessment of the SPR binding data reveals that the 5–10-fold difference in the equilibrium binding constants between GPIHBP11–131 and GPIHBP134–131 could be accounted for by the differences in their association rate constants: kon = 1 . 04 × 106 M–1s–1 for GPIHBP11–131versus a kon = 0 . 10 × 106 M–1s–1 for GPIHBP134–131 ( Table 1 ) . In contrast , the stabilities of the GPIHBP1•CTD complexes with GPIHBP11–131 and GPIHBP134–131 were essentially identical; both displayed koff values of 0 . 1 s–1 . Removal of the single N-linked glycosylation in GPIHBP11–131 had no significant impact on the kinetic rate constants ( Table 1 ) . As these analyses were conducted with the R38G mutation in GPIHBP1 ( which was introduced to optimize purification yields ) , we tested whether this mutation affects the binding kinetics between LPL and GPIHBP1 . To liberate intact , wild-type GPIHBP11–131 , we performed a partial cleavage of 7 mg RS-DIII-ent-GPIHBP1 with 0 . 05 U enterokinase/mg protein , which resulted in 70% cleavage of the fusion protein . A sufficient amount of this full-length GPIHBP11–131 was purified by ion-exchange chromatography to allow subsequent kinetic analyses by SPR . The kinetics for the interactions between mAb 5D2-captured CTD or LPL and wild-type GPIHBP11–131 were similar to those measured with GPIHBP11–131/R38G ( Table 1 ) . Thus , the R38G mutation does not significantly affect the properties of the GPIHBP1 interaction with LPL . A similar kinetic assessment for the binding of LPL homodimers by multi-cycle titration was , however , inherently challenging because the conditions required to regenerate mAb 5D2 between cycles progressively reduced the capturing capacity of the sensor chip . To minimize this confounding factor , we performed a single-cycle kinetic titration of the interaction between GPIHBP1 and full-length LPL dimers captured by mAb 5D2 . To be confident that the captured LPL was in a properly folded and homodimeric state , we initially performed a kinetic titration with two LPL-specific monoclonal antibodies ( mAb 4-1a , which binds within residues 5–25 in LPL [Bensadoun et al . , 2014] and mAb 5D2 , which binds to residues 380–400 in the CTD of LPL [Chang et al . , 1998] ) . As shown in Figure 4A , both antibodies bound immobilized LPL when analyzed by a single-cycle titration protocol . Calculation of their binding capacities [Rmax = 200 RU for mAb 4-1a ( 1 . 3 fmols/mm2 ) and Rmax = 309 RU for mAb 5D2 ( 2 . 1 fmols/mm2 ) ] revealed molar surface-binding capacities that were similar to the surface density of the captured LPL homodimer ( R900sec = 203 RU [2 . 0 fmols/mm2] ) . Because mAb 5D2 is used as both capture and detection agent in this experiment , our data suggest that the majority of the captured LPL on the sensor chip was in the form of homodimers . The single-cycle kinetic titrations revealed fast kon values for GPIHBP1•LPL interactions . The koff values for the LPL complexes with GPIHBP11–131 and GPIHBP134–131 were 5-fold slower than those for the corresponding CTD interaction , which indicates that GPIHBP1 forms a tighter complex with full-length LPL ( Table 1 , Figure 4B and 4C ) . To supplement the real-time binding kinetics recorded by SPR , we measured the equilibrium binding constants for the interaction between GPIHBP11–131/R38G and LPL in solution by microscale thermophoresis ( MST ) with a Monolith NT . 115 ( NanoTemper Technologies GmbH; Germany ) . In these studies , 5 nM Alexa-647–labeled GPIHBP11–131/R38G or GPIHBP134–131/R38G were incubated with increasing concentrations of LPL2 ( 10 pM to 350 nM ) . The binding isotherms were then calculated from the shifts in the thermophoresis of the fluorophore ( Figure 5 ) . With this experimental approach , we determined a KD of 5 . 7 nM for the LPL•GPIHBP11–131/R38G interaction and a KD of 147 nM for the GPIHBP134–131/R38G interaction . All things considered , we find that data with this solution-based assay aligns excellently with the binding affinities determined kinetically by SPR . 10 . 7554/eLife . 12095 . 013Figure 5 . Equilibrium binding constants of the GPIHBP1•LPL interaction by microscale thermophoresis . The microscale thermophoresis signals for the interaction of LPL with 5 nM Alexa Flour-647–labeled GPIHBP11–131/R38G ( panel A ) and GPIHBP134–131/R38G ( panel B ) were recorded in quadruplicates for two-fold dilution series of unlabeled LPL2 ( 10 pM to 350 nM ) . The mean values and standard deviation for the thermophoresis are shown as well as a fitting to a 1:1 binding model ( software supplied with Monolith NT . 115 ) . LPL , lipoprotein lipase . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 013 The impact of GPIHBP1 on the spontaneous unfolding of LPL was subsequently addressed by HDX-MS . This was accomplished by incubating LPL in the presence of GPIHBP11–131 , GPIHBP11–33 , or GPIHBP134–131 for 45 min at 25°C . We assessed the deuterium uptake after 10-s labeling with D2O . GPIHBP11–131 clearly inhibited LPL unfolding ( Figure 6C ) . This protective effect was predominantly due to the acidic domain because the GPIHBP11–33 peptide alone also attenuated LPL unfolding . A distinct but much less pronounced contribution was provided by GPIHBP134–131 on both unfolding rates and preservation of triolein hydrolase and esterase activity of LPL ( Table 2 ) . 10 . 7554/eLife . 12095 . 015Table 2 . Preservation of LPL structure and activity by GPIHBP1 binding . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 015Added ligandUnfolding of NTD in LPL ( % ) aLoss of triolein hydrolase activity ( % ) bLoss of esterase activity ( % ) bGPIHBP11–131/R38G32 . 6 ± 5 . 728 . 7 ± 1 . 511 . 4 ± 6 . 7GPIHBP134–131/R38G79 . 2 ± 4 . 251 . 5 ± 1 . 334 . 7 ± 8 . 4GPIHBP11–3343 . 0 ± 5 . 14 . 2 ± 1 . 8b9 . 8 ± 6 . 9bNone100100100aInhibition of the spontaneous decay of LPL by GPIHBP11–131 , GPIHBP134–131 , or GPIHBP11–33 was measured after a 45-min incubation at 25°C . Equimolar amounts of GPIHBP1 and LPL homodimers were present during the incubation step . bTriolein hydrolase activity was measured with a [3H]triolein substrate; esterase activity was measured with a soluble fluorescent substrate . Activity assays were performed on LPL samples that were subjected to the same pre-incubation conditions used for the HDX-MS experimentsa except that GPIHBP11–33was present in 5-fold molar excess compared to LPL . The protection by GPIHBP1 variants is shown relative to the decay of LPL after a 45-min incubation without any added binding partner ( defined as 100% ) . Measurements were performed in triplicate . To map defined binding sites for GPIHBP1 on LPL , we used zero-length covalent cross-linking with N-ethyl-N′-[3-diethylamino ) propyl]-carbodiimide ( EDC ) , which forms an isopeptide bond by chemical condensation of carboxylates and primary amino groups that are in close spatial proximity . For these studies , we formed GPIHBP1•LPL complexes by incubating a high concentration of LPL with a twofold molar excess of GPIHBP11–131 or GPIHBP134–131 ( to saturate binding sites on LPL ) . Adding 10 mM EDC efficiently cross-linked LPL•GPIHBP11–131 in a 1:1 complex , consuming the majority of the LPL ( Figure 7 , lanes 5 & 11 ) . In contrast , no covalent adducts were formed in the samples containing GPIHBP11–131 alone , LPL alone , or a mixture of LPL and GPIHBP134–131 ( Figure 7 , lanes 3 , 4 , 6 & 10 ) . Adding EDC to LPL in the presence of a 10-fold molar excess of GPIHBP11–33 also led to the formation of a 1:1 complex with no evidence of higher-order adducts ( Figure 7 , lane 9 ) . These studies imply that each LPL molecule interacts with only a single acidic domain peptide . 10 . 7554/eLife . 12095 . 016Figure 7 . Zero-length cross-linking of GPIHBP11–131 and GPIHBP11–33 to bovine LPL . Panel A , left shows a Coomassie Blue–stained 12% polyacrylamide gel after SDS-PAGE analysis of reduced and alkylated samples representing various combinations of 1 . 5 µM LPL2 and 7 µM GPIHBP1 variants subjected to EDC cross-linking . Lane 2 shows LPL GPIHBP11–131 before cross-linking . Lanes 3–6 show samples after EDC cross-linking: GPIHBP11–131 ( lane 3 ) ; LPL ( lane 4 ) , LPL GPIHBP11–131 ( lane 5 ) , and LPL GPIHBP134–131 ( lane 6 ) . The covalently bound conjugate representing LPL•GPIHBP11–131 is marked by an asterisk . Right panel shows a Coomassie Blue–stained 4–12% gradient polyacrylamide gel after analysis of EDC cross-linked 3 µM LPL2 alone ( lane 8 ) or in the presence of 15 µM GPIHBP11–33 ( lane 9 ) ; 15 µM GPIHBP134–131 ( lane 10 ) ; and 15 µM GPIHBP11–131 ( lane 11 ) . The covalent conjugates representing LPL•GPIHBP11–131 and LPL•GPIHBP11–33 are indicated by an asterisk and a solid dot , respectively . Molecular weight markers are shown in lanes 1 , 7 & 12 . Panel B shows a model of human LPL highlighting the cross-linking sites in GPIHBP1 that were identified by MS ( asterisks ) . Areas that have been assigned as potential interaction sites for GPIHBP1 by HDX-MS are shown in green ( for the acidic domain of GPIHBP1 ) and blue ( for the LU domain of GPIHBP1 ) . The position of the interdomain interface in LPL between the NTD and CTD is marked by a dashed line , and three residues within this interface linked to familial chylomicronemia when mutated ( S259R , G409R , and E410V ) are shown by gray numbers . Basic residues of the heparin-binding site in the catalytic domain of LPL ( R279 , K280 , R282 ) are shown as sticks . Note , bovine LPL contains two additional residues compared with human LPL , for example Lys296 in human LPL is equivalent to Lys298 in bovine LPL . CTD , C-terminal domain; EDC , N-ethyl-N′-[3-diethylamino ) propyl]-carbodiimide; HDX-MS , hydrogen–deuterium exchange mass spectrometry; LPL , lipoprotein lipase; MS , mass spectrometry; NTD , N-terminal domain; SDS-PAGE , sodium dodecyl sulfate polyacrylamide gel electrophoresis . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 01610 . 7554/eLife . 12095 . 017Figure 7—figure supplement 1 . Fragment spectra identifying EDC-mediated inter-domain cross-links in mature LPL•GPIHBP11–131 complexes . Panel A shows the fragment spectra produced for the EDC cross-linked peptide between residues 1–33 in GPIHBP1 and residues 295–299 ( VARKR ) in bovine LPL . Panel B shows the fragment spectra produced for the cross-linked peptide between residues 1–33 in GPIHBP1 and residues 416–422 ( KVIFCSR ) in bovine LPL . Fragments derived from residues 1–33 of GPIHBP1 are designated ( A ) and peptides from LPL are designated ( B ) . The blue peaks correspond to y-fragment ions from either peptide; green peaks correspond to b-fragments ions from either peptide; red peaks represent unassigned peaks; purple peaks correspond to loss of NH3 or H2O ( not labeled ) or ions that have undergone two fragmentation reactions . Peaks corresponding to cross-linked peptide fragments are assigned by an × along with the two peptide fragments ( e . g . DEDH×KR ) . EDC , N-ethyl-N′-[3-diethylamino ) propyl]-carbodiimide; LPL , lipoprotein lipase . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 01710 . 7554/eLife . 12095 . 018Figure 7—figure supplement 2 . SDS-PAGE analysis of EDC/NHS cross-linked LPL•GPIHBP1 complexes . The cross-linking efficiencies between LPL and GPIHBP134–131 or GPIHBP11–131 are revealed by a Coomassie Blue–stained 12% SDS-polyacrylamide gel . The samples were cross-linked for 4 hr by 5 mM EDC and NHS in the following format: 3 µM LPL2 alone ( lane 3 ) or in the presence of 15 µM GPIHBP134–131 ( lane 4 ) or 15 µM GPIHBP11–131 ( lane 5 ) . LPL without EDC/NHS is shown in lane 2 . Molecular mass markers are in lane 1 . The asterisk shows a covalent LPL•GPIHBP134–131 conjugate . Using these cross-linking conditions , we observe a low level of covalently-linked LPL dimers ( lane 3 ) ; the amount of these LPL dimers is reduced by the presence of GPIHBP134–131 or GPIHBP11–131 ( lane 4 & lane 5 ) , likely reflecting the engagement of LPL in interactions with these ligands . EDC , N-ethyl-N′-[3-diethylamino ) propyl]-carbodiimide; LPL , lipoprotein lipase; NHS , N-hydroxysuccinimide; SDS-PAGE , sodium dodecyl sulfate polyacrylamide gel electrophoresis . DOI: http://dx . doi . org/10 . 7554/eLife . 12095 . 018 To identify the specific cross-linked sites responsible for the formation of the covalent LPL•GPIHBP1 adduct , we performed an in-gel trypsin digestion of the complex that had been separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) ( Figure 7 , lane 5 ) . The extracted tryptic peptides were analyzed by a high-resolution Q-Exactive HF mass spectrometer , and cross-linked peptides were identified by their parent ion mass as well as subsequent HCD fragment spectra ( Figure 7—figure supplement 1 ) . All but one of the five identified cross-links was established between LPL and GPIHBP1’s acidic domain , consistent with the abundance of EDC-reactive carboxyl groups in the acidic domain . The corresponding cross-linking sites on LPL included Lys298 in the catalytic domain and Lys416 , Lys424 , and Lys430 in the CTD of LPL ( Figure 7B ) . The one cross-linked peptide identified outside of the acidic domain occurred between residues 100–125 ( most likely Glu102 ) in the LU-domain of GPIHBP1 and residues 423–430 of LPL . We did not detect cross-links between GPIHBP134–131 and LPL using 10 mM EDC for 90 min ( Figure 7 , lanes 6 & 10 ) ; however , we did observe such cross-links ( Figure 7—figure supplement 2 ) if we drove the conjugation chemistry to higher yields by converting the o-acylisourea EDC intermediate to a more stable amine-reactive derivative ( N-hydroxysuccinimide ( NHS ) -ester ) .
The interaction between LPL and GPIHBP1 is essential for lipolysis of TRLs along the capillary lumen ( Beigneux et al . , 2007; Davies et al . , 2010; Goulbourne et al . , 2014 ) . In the absence of functional GPIHBP1 , LPL is mislocalized within the interstitial spaces , leading to severe hypertriglyceridemia and reduced delivery of lipid nutrients to parenchymal cells ( Goulbourne et al . , 2014; Young and Zechner , 2013 ) . LPL or GPIHBP1 mutations that prevent LPL•GPIHBP1 binding or abolish LPL catalytic activity result in familial chylomicronemia in humans ( Buonuomo et al . , 2015; Gin et al . , 2012; Henderson et al . , 1996; Plengpanich et al . , 2014; Rios et al . , 2012; Voss et al . , 2011 ) . While the physiologic function of GPIHBP1 has gradually come into focus , our understanding of LPL•GPIHBP1 interactions was negligible . No three-dimensional structures had been determined for LPL or GPIHBP1 , almost certainly because of the low stability of LPL and earlier difficulties in preparing pure recombinant GPIHBP1 free of protein dimers and multimers ( Beigneux et al . , 2015 ) . In the current study , we eliminated one of the roadblocks to progress by developing protocols for expressing and purifying human GPIHBP1 . Our approach took advantage of Drosophila S2 cells as host cells for heterologous expression . Other LU domain–containing proteins have been expressed with this system ( Gårdsvoll et al . , 2007; Gårdsvoll et al . , 2004 ) , and the high quality of those protein preparations are highlighted by both the structures solved by X-ray crystallography ( Lin et al . , 2010; Llinas et al . , 2005; Xu et al . , 2012; Zhao et al . , 2015 ) and the structure–function insights gained by biophysical approaches ( HDX-MS , SAXS , and SPR ) ( Gårdsvoll et al . , 2006; Jørgensen et al . , 2004; Mertens et al . , 2012 ) . In the case of human GPIHBP1 , we were able to prepare large quantities of highly purified , monomeric proteins for full-length GPIHBP11–131 as well as a truncated GPIHBP1 lacking the acidic domain ( GPIHBP134–131 ) . The latter protein was generated by limited proteolysis of full-length GPIHBP1 . With the availability of homogeneous preparations of catalytically active LPL and pure , monomeric GPIHBP1 , we uncovered several novel properties of the GPIHBP1•LPL interaction , all relevant to the physiology of intravascular TRL processing . We found: ( 1 ) that the N-terminal acidic domain of GPIHBP1 is intrinsically disordered; ( 2 ) that the acidic domain has a major effect on the association rate constant for the GPIHBP1•LPL interaction; ( 3 ) that the time-dependent decay of LPL activity is associated with a global unfolding of LPL’s catalytic domain; ( 4 ) that the acidic domain of GPIHBP1 preserves the catalytic activity of LPL by mitigating global unfolding of the catalytic domain; and ( 5 ) that two discrete interaction sites in the LPL•GPIHBP1 complex cooperate to promote ligand binding and stabilization of LPL catalytic activity . Our findings have allowed us to conceptualize a model for unoccupied GPIHBP1 in which the N-terminal acidic domain is intrinsically disordered and the remainder of the protein ( i . e . GPIHBP134–131 ) adopts the prototypical three-finger-fold characteristic of LU domain proteins ( Figure 1A ) . This model was initially suggested by the observation that full-length GPIHBP1 ( GPIHBP11–131 ) exhibits an unexpectedly large hydrodynamic volume ( as judged by its elution profile in size-exclusion chromatography [Figure 1C] ) , whereas a truncated GPIHBP1 lacking the acidic domain ( GPIHBP134–131 ) elutes at the expected position for a folded globular protein . The atypical elution profile for the full-length GPIHBP1 is indicative of a large Stokes radius and is one of the hallmarks of intrinsically disordered proteins ( Uversky , 2012 ) . Further evidence for the disordered nature of the acidic domain is provided by its highly dynamic state as measured by HDX-MS . Our model offers potential insights into the interplay between GPIHBP1 and LPL during LPL mobilization and TRL processing in vivo . In GPIHBP1-deficient mice , the LPL in tissues is mislocalized to the interstitial spaces and never reaches the luminal surface of capillary endothelial cells . It is noteworthy that the LPL in GPIHBP1-deficient mice remains sequestered in the interstitial space , presumably bound to negatively-charged heparin sulfate proteoglycans ( HSPGs ) ( Davies et al . , 2010 ) . This interaction is thought to be driven by electrostatic interactions and is characterized by fast kinetic rate constants ( Lookene et al . , 1996 ) , presumably creating a dynamic reservoir of LPL within the interstitial spaces . In wild-type mice , newly secreted LPL likely binds to the same HSPGs but then moves to GPIHBP1 on capillary endothelial cells . We propose that the intrinsically disordered and markedly acidic N-terminal extension of GPIHBP1 plays an important role in mobilizing HSPG-bound LPL within the interstitial spaces through long-ranged electrostatic interactions , thereby driving LPL into association with endothelial cells . This model for LPL mobilization by GPIHBP1 is consistent with the kinetic rate constants that we determined for GPIHBP1•LPL interactions , which are 10-fold faster for intact GPIHBP11–131 than for GPIHBP134–131 lacking the acidic domain . Since the difference in kon for GPIHBP11–131 and GPIHBP134–131 is observed for both intact LPL and LPL’s CTD alone , we propose that an electrostatic 'encounter complex' is initially established between GPIHBP1’s acidic domain and the basic residues in LPL’s CTD . This encounter complex then guides the maturation of the interaction , facilitating a tighter binding interface between GPIHBP1’s LU domain and residues 402–419 in LPL ( Figure 2 ) . The cross-linking that we documented between residues 1–33 of intact GPIHBP11–131 and Lys416 , Lys424 , and Lys430 in the CTD of LPL is consistent with this initial encounter/maturation model . The electrostatic component of this model is in accordance with a previous study ( Reimund et al . , 2015 ) . The existence of a dual binding mechanism is also consistent with the available mutagenesis data . First , the importance of the positively charged heparin-binding sequences in LPL’s CTD has been demonstrated by reduced binding of hLPLK403A;R405A;K407A;K413A;K414A to GPIHBP1 ( Gin et al . , 2008 ) . Second , there is ample evidence for the involvement of additional CTD sequences—apart from positively charged residues—in the binding of LPL to the LU-domain in GPIHBP1 . For example , two missense mutations in LPL ( C418Y and E421K ) , first identified in patients with chylomicronemia , have no effect on heparin binding but abolish binding to GPIHBP1 ( Henderson et al . , 1998; Henderson et al . , 1996; Voss et al . , 2011 ) . Lending support to this two-step binding mechanism is the fact that the acidic domain plays little or no role in the stability of established GPIHBP1•LPL complexes . The koff values for GPIHBP11–131 and GPIHBP134–131 are comparable , regardless of whether the binding is to LPL’s CTD ( koff = 0 . 1 s–1 ) or to full-length LPL ( koff = 0 . 02 s–1 ) . A different two-site interaction model between GPIHBP1 and LPL was recently proposed ( Reimund et al . , 2015 ) , which suggested that a single molecule of GPIHBP1 binds two LPL homodimers . This conclusion was based on SPR studies in which GPIHBP1 ( from the medium of transfected CHO cells ) had been captured onto chips coated with the anti-GPIHBP1–specific mAb 11A12 . To obtain reliable fits for the LPL-binding profiles recorded by SPR and reduce nonspecific binding of LPL , these authors performed their analyses at 4°C and in the presence of 0 . 4–0 . 6 M NaCl . In the current study , the SPR data were obtained under more physiologic conditions ( 20°C in 0 . 15 M NaCl ) , and the binding profiles fit well to a simple 1:1 interaction model ( i . e . GPIHBP1+LPL2⇆ GPIHBP1•LPL2 ) . A likely explanation for the discrepancy between the two studies is the different designs of the SPR studies . The current study measured the binding of purified , monomeric GPIHBP1 in solution to purified LPL that had been captured by mAb 5D2 thereby avoiding the inherent difficulties in having LPL as a soluble analyte . The earlier study measured the interaction between LPL in solution to mouse GPIHBP1 ( from the medium of transfected CHO cells ) that had been captured on mAb 11A12 . We now know the GPIHBP1 secreted from transfected CHO-cells contains significant amounts of dimers and multimers ( Beigneux et al . , 2015 ) , a factor that would further complicate the interpretation of these SPR data . An intriguing finding in the current study was the observation that LPL undergoes a time-dependent global unfolding of its catalytic N-terminal domain , as judged by the loss of stable secondary structure in HDX-MS studies ( Figure 7 ) . This transition into a pre-molten globule–like conformation of LPL’s catalytic domain provides a plausible structural explanation for the time-dependent decline in LPL catalytic activity at room temperature ( Osborne et al . , 1985 ) . It is noteworthy , however , that unfolding of the catalytic domain in LPL proceeds with only one-half the rate of the parallel loss of catalytic activity . That observation is consistent with a model where the unfolding of one LPL molecule in the homodimer would lead to the inactivation of both subunits . From our HDX-MS analyses of GPIHBP1•LPL complexes ( Figure 2 ) , it is apparent that only one region in LPL responds to the presence of the acidic domain GPIHBP11–33 , and that is located at the interface between the CTD and the catalytic domain ( represented by the peptide spanning residues 279–290 ) ( Figure 7B ) . The involvement of this 'interdomain interface' of LPL in binding GPIHBP11–33 was bolstered by zero-length cross–linking studies . The interaction between LPL Lys298 ( in close spatial proximity to peptide 279–290 ) and GPIHBP1’s acidic domain was covalently trapped by EDC ( Figure 7B ) . The fact that the acidic domain interacts with the domain interface of LPL in the setting of a mature GPIHBP1•LPL complex likely carries functional implications . Indeed , the stabilization of the catalytic domain of LPL by GPIHBP11–131 was primarily mediated by GPIHBP11–33 ( Table 1 ) . We suspect that the stabilization of LPL’s 'interdomain interface' by GPIHBP1’s acidic domain serves to limit protein dissociation/unfolding of the partner monomers of an LPL homodimer . Genetic evidence provides circumstantial support for the importance of a properly assembled interdomain interface for LPL activity . Certain homozygous missense mutations that are associated with reduced LPL activity and chylomicronemia [LPLE410V , LPLG409R , and LPLS259R in humans; LPLG412R in cats ) ( Foubert et al . , 1997; Ginzinger et al . , 1996; Kassner et al . , 2015; Previato et al . , 1994 ) ] are located at the interdomain interface in close spatial proximity to the proposed binding site in LPL279–290 for GPIHBP11–33 ( Figure 7B ) . All mutations alter the electrostatics of the interface . In future studies , it will also be very interesting to investigate if the accelerated inactivation of LPL by ANGPTL4 ( Sukonina et al . , 2006 ) involves this interdomain region and the extent to which GPIHBP1 protects LPL from ANGTPL4-mediated inactivation . From a practical point of view , our discovery that the acidic domain stabilizes the conformation of LPL by mitigating unfolding could prove to be helpful in future efforts to define the structure of LPL by X-ray crystallography . We suspect that incubating LPL with peptides derived from GPIHBP1’s acidic domain could promote stabilization of LPL , increase its conformational homogeneity , and enhance the likelihood of growing well-diffracting crystals for X-ray structure determination .
Bovine LPL was purified from fresh bovine milk by heparin-Sepharose , hydroxyapatite , and Superdex HR200 size-exclusion chromatography as described ( Cheng et al . , 1985 ) . Fractions containing dimeric LPL ( LPL2 ) were precipitated with 3 . 6 M NH4SO4 and the pellet dissolved in 5 mM phosphate buffer ( pH 6 . 5 ) containing 40% ( v/v ) glycerol . Small aliquots were stored at –80°C until use . Recombinant enterokinase ( 1 U/µl ) produced in Pichia pastoris ( EKMax ) was purchased from Invitrogen ( Carlsbad , CA ) . Monoclonal antibodies against human uPAR ( R2 and R24 ) and human LPL ( 5D2 and 4-1A ) were produced and characterized as described ( Bensadoun et al . , 2014; Chang et al . , 1998; Gårdsvoll et al . , 2011 ) . A synthetic 33-mer peptide ( GPIHBP11–33 ) representing the N-terminal acidic domain of GPIHBP1 ( QTQQEEEEEDEDHGPDDYDEEEDEVEEEETNR ) was obtained at a purity of >95% from TAG-Copenhagen A/S ( Copenhagen , Denmark ) . A truncated version of human GPIHBP1 ( residues 1–131 ) , lacking the C-terminal signal peptide that normally triggers the covalent addition of a glycolipid anchor , was expressed by ( and secreted from ) Drosophila S2 cells as a fusion protein with uPAR domain III ( RS-DIII-ent-GPIHBP1 , Figure 1—figure supplement 1A ) . This strategy has been used for expressing other LU-domain proteins ( Gårdsvoll et al . , 2007; Gårdsvoll et al . , 2013; Hansen et al . , 2004 ) . The N-terminal uPAR DIII serves the dual role of both the detection and the purification tag , and the DDDDK motif in the linker region allows excision of GPIHBP1 by enterokinase cleavage while preserving the original N-terminus of GPIHBP1 . Using this strategy , we also expressed the mutants RS-DIII-ent-GPIHBP1W89S and RS-DIIIR281G-ent-GPIHBP1R38G . The latter construct was designed to improve the overall yield of purified intact GPIHBP1 after enterokinase cleavage . A similar platform was employed to express the CTD of human LPL ( residues 313–448 ) using constructs RS-DIII-ent-CTD and RS-DIII-ent-CTDC418Y ( Figure 1—figure supplement 1B ) . After 7 days of induction with 0 . 5 mM CuSO4 , the medium from the Drosophila S2 cells was harvested and supplemented with 0 . 1 volume of 1 M Tris/HCl ( pH 8 . 0 ) containing 0 . 2 M ethylenediaminetetraacetic acid ( EDTA ) and 0 . 1% ( w/v ) NaN3 and 0 . 005 volume of 200 mM PMSF in DMSO . The various RS-DIII-ent-GPIHBP1 proteins were purified from the medium of transfected cells by immunoaffinity chromatography with an anti-uPAR mAb R2 column with 0 . 1 M CH3COOH , 0 . 5 M NaCl ( pH 2 . 5 ) as the eluent . The purified proteins were exchanged into 20 mM NaH2PO4 , 150 mM NaCl ( pH 7 . 2 ) and stored at –80°C . The RS-DIII-ent-CTD constructs were affinity purified with an anti-uPAR mAb R24 column ( Gårdsvoll et al . , 2011 ) and eluted with 0 . 1 M CH3COOH , 0 . 5 M NaCl ( pH 2 . 5 ) containing 20% ( v/v ) glycerol and then exchanged into 20 mM NaH2PO4 , 150 mM NaCl ( pH 7 . 2 ) containing 20% ( v/v ) glycerol to prevent precipitation . Average yields of the fusion proteins were 2–5 mg/l . The DIII-tag was removed from the purified fusion proteins by adding 0 . 1 U enterokinase/mg and incubating at 37°C . An equal amount of enterokinase was added 6 hr later , and the incubation was continued for a total of 24 hr . This digest was dialyzed against 50 mM CH3COOH ( pH 4 . 5 ) . The excised GPIHBP1 was purified by cation-exchange chromatography with a 5-ml HiTrap SP FF ( GE Healthcare; Uppsala , Sweden ) and a 35-ml linear NaCl gradient ( from 0 to 1 . 0 M ) in 50 mM CH3COOH ( pH 4 . 5 ) ( Figure 1—figure supplement 2A ) . Relevant fractions were pooled and buffer-exchanged into 20 mM NaH2PO4 , 150 mM NaCl ( pH 7 . 2 ) before conducting a final purification step by size-exclusion chromatography on a Superdex HR75 column in the same buffer ( Figure 1C ) . To minimize confounding effects resulting from the dextran surface of the sensor chip on the real-time kinetics recorded for the LPL•GPIHBP1 interaction , we used a CM4 sensor chip with a low degree of carboxylation to immobilize the LPL-specific monoclonal antibody 5D2 ( Chang et al . , 1998 ) . As a further precaution , we used 1 M ethylenediamine rather than ethanolamine to block excess active NHS esters , which reduces the charge effects from the remaining unmodified carboxylates on the matrix ( Glaser et al . , 2014 ) . Covalent immobilization of mAb 5D2 was accomplished by injecting 5 µg/ml 5D2 dissolved in 10 mM sodium acetate ( pH 5 . 0 ) over a CM4 chip that had been pre-activated with NHS/EDC ( N-ethyl-N´-[3-diethylamino ) propyl]-carbodiimide ) , aiming at a surface density of 1500 resonance units ( RU ) corresponding to 10 fmols/mm2 . After a 100-s loading pulse with 100 nM recombinant LPL CTD313–448 at 20 µl/min , real-time interactions between 5D2-captured CTDs ( ~100–200 RU; 6–12 fmol/mm2 ) and serial twofold dilutions of purified human GPIHBP1 were measured from 8 nM to 4 µM at 20°C using 10 mM HEPES , 150 mM NaCl , 3 mM EDTA ( pH 7 . 4 ) containing 0 . 05% ( v/v ) surfactant P20 as running buffer . To be confident of a high reproducibility , at least one GPIHBP1 concentration was re-tested at the end of each experiment . Between cycles , mAb 5D2 was regenerated with two consecutive 10-µl injections of 0 . 1 M acetic acid/HCl ( pH 2 . 5 ) in 0 . 5 M NaCl and 20 mM H3PO4 . The immobilized mAb 5D2 tolerated repetitive regenerations without significant decline of its capturing capacity . After double-buffer referencing of the recorded real-time interaction analyses , the kinetic rate constants ( kon and koff ) were derived by global non-linear regression fitting of the association and dissociation phases to a simple bimolecular interaction model , including correction for mass transport limitations assuming pseudo–first-order reaction conditions with BIA evaluation 4 . 1 software ( Biacore , Uppsala , Sweden ) . In some cases , the equilibrium binding constants KD and Rmax were also calculated separately from the corresponding equilibrium binding isotherms by nonlinear curve fitting assuming saturation of a single binding site:Req= ( Rmax[GPIHBP1] ) / ( Kd+[GPIHBP1] ) , where Req is the binding level at equilibrium , and Rmax is the binding capacity of the chip . To enable efficient capturing of intact dimeric bovine LPL ( LPL2 ) on immobilized mAb 5D2 , the purified LPL2 was diluted 500–1000 fold to 200 nM in 10 mM HEPES ( pH 7 . 4 ) , 150 mM NaCl , 4 mM CaCl2 , 0 . 05% ( v/v ) surfactant P20 supplemented with 0 . 2 mg/ml bovine serum albumin ( BSA ) and 1% ( w/v ) carboxymethyl dextran to stabilize LPL and avoid non-specific adsorption to the microfluidics during loading . The running buffer was equivalent to the loading buffer , except that no carboxymethyl dextran was added . Regeneration of the capturing mAb was accomplished by two consecutive injections of 10 µl of 20 mM glycine/HCl ( pH 2 . 5 ) and 10 µl of 5% ( v/v ) HCOOH-containing 0 . 5 M NaCl . To minimize deterioration from repetitive regenerations , single-cycle kinetic titration ( Karlsson et al . , 2006 ) of LPL•GPIHBP1 interactions were recorded during five consecutive injections of 20 µl of two-fold dilutions of purified GPIHBP1 at 20°C . After double-buffer referencing of the data , the corresponding kinetic rate constants were derived by fitting the data to a simple bimolecular interaction model using the mathematic model developed for single-cycle kinetics ( T200 Evaluation Software 2 . 0 , GE Healthcare Life Science , Uppsala , Sweden ) . To determine solution equilibrium binding constants between LPL and Alexa Flour-647–labeled versions of GPIHBP11–131/R38G and GPIHBP134–131/R38G , we used the MST technology ( Jerabek-Willemsen et al . , 2014 ) . Purified GPIHBP11–131/R38G and GPIHBP134–131/R38G preparations were labeled for 30 min at 37°C with Alexa Flour-647 NHS ester ( Thermo Fisher Scientific ) at a molar ratio of 1:3 in phosphate-buffered saline ( pH 7 . 4 ) ; these conditions favor modification of the N-terminal α-amino group . The reaction was terminated by adding 10 mM ethanolamine , and the proteins were desalted on a PD-10 column ( GE Healthcare , Uppsala , Sweden ) . The average degree of labeling was 1 . 3 flurophore/protein for both GPIHBP11–131/R38G and GPIHBP134–131/R38G; both labeled proteins remained monomeric as judged by analytical size-exclusion chromatography on a 5/150 Superdex 75 column ( GE Healthcare , Uppsala , Sweden ) . The equilibrium binding between 5 nM Alexa Flour-647–labeled GPIHBP1 and LPL were calculated from the change in thermophoresis ( ΔFnorm = FHot/Fcold ) measured with a Monolith NT . 115 ( NanoTemper Technologies GmbH ) after adding increasing concentrations of non-fluorescent LPL . A two-fold dilution series ranging from 10 pM to 350 nM LPL2 was prepared in 10 mM HEPES ( pH 7 . 4 ) , 150 mM NaCl , 4 mM CaCl2 , 0 . 05% ( v/v ) surfactant P20 , and 1 . 0 mg/ml BSA . Samples were loaded into low-binding hydrophilic capillary tubes and the thermophoresis signals were measured at 22°C with a light-emitting diode ( LED ) power of 80% and an infrared ( IR ) laser power of 100% . Time-dependent inactivation profiles for LPL were determined by incubating 2 µM purified LPL2 alone or in the presence of 2 µM GPIHBP11–131 , 2 µM GPIHBP134–131 , or 10 µM GPIHBP11–33 for various times at 25°C in 10 mM Na2HPO4 , 150 mM NaCl ( pH 7 . 4 ) . The temperature-induced unfolding was quenched by adding ice-cold 100 mM Tris ( pH 7 . 8 ) containing 5 mM deoxycholic acid ( DOC ) and 0 . 1 mM sodium dodecyl sulfate ( SDS ) ( DOC/SDS buffer ) . Esterase activities were analyzed by mixing 5 μl of LPL diluted 100-fold in DOC/SDS buffer with 95 μl DGGR assay buffer to a final concentration of 50 mM Tris , 50 μM 1 , 2-o-dilauryl-rac-glycero glutaric acid- ( 6'-methylresorufin ) ester ( DGGR ) , 120 mM NaCl , 10 mg/ml BSA , 0 . 5% Triton X-100 ( v/v ) , pH 7 . 4 . Ester hydrolysis was determined by measuring the linear increase of resorufin fluorescence at λex 530 nm , λem 590 nm during the initial 5 min . Lipase activities were determined by adding 5 μl of LPL diluted 1000-fold in DOC/SDS to 195 μl of incubation mixtures with Intralipid containing [3H] triolein ( Larsson et al . , 2013 ) . All hydrogen–deuterium exchange reactions were performed at 25°C and 300 RPM mixing , using 10 mM Na2HPO4 , 150 mM NaCl buffers in either H2O or D2O , adjusted to pH 7 . 4 and pD 7 . 4 ( pHread = 7 . 0 ) , respectively . The following protein solutions were made with the H2O buffer: 5 µM LPL2 , 5 µM GPIHBP11–131/R38G , 5 µM LPL2 5 µM GPIHBP11–131/R38G , 5 µM LPL2 25 µM GPIHBP11–33 . Most solutions were pre-incubated for 15 min before deuterium labeling to promote efficient complex formation . Samples containing exclusively LPL2 were not pre-incubated because of concerns about the inherent instability of LPL homodimers . Isotopic labeling was initiated by adding D2O buffer to the protein solutions to a final concentration of 70% D2O ( vol/vol ) . After 10- , 100- or 1 , 000-sec , aliquots were collected from the labeling solutions . These were mixed with 1 volume ice-cold quenching buffer ( 100 mM Na2HPO4 , 0 . 8 M TCEP , 2 M urea in H2O , pH 2 . 5 ) . Quenched samples were placed in an ice bath for 2 min to reduce disulfide bonds and subsequently snap-frozen in liquid nitrogen . Full deuteration controls were prepared by ( 1 ) diluting LPL and GPIHBP1 to 10 µM in 10 mM Na2HPO4 , 150 mM NaCl , 2 M urea , 70% ( vol/vol ) D2O , pD 7 . 4; ( 2 ) incubating samples for 48 hr at 37°C; and ( 3 ) quenching as described above without urea to achieve an identical solvent composition in the quenched samples . Labeling was performed in triplicate for each sample combination . Buffers and labeling conditions were identical to those described previously . The following protein solutions were prepared in H2O buffer: 5 µM LPL2 , 5 µM LPL2 5 µM GPIHBP11–131/R38G , 5 µM LPL2 5 µM GPIHBP11–33 , and 5 µM LPL2 5 µM GPIHBP134–131/R38G . Solutions were incubated for 15 , 30 , 45 , 60 , 90 , and 120 min , diluted to 70% D2O buffer , allowed to exchange for 10 s , quenched by adding 1:1 ice-cold quench buffer , incubated for 2 min in an ice bath , and then snap frozen . Labeling was performed in triplicate for each sample combination incubated at 45 min . Quenched and reduced samples were analyzed with a nanoACQUITY UPLC reversed-phased chromatographic system equipped with HDX technology ( Waters , Milford , MA ) coupled to a Synapt G2 electrospray ionization mass spectrometer ( Waters ) . Desalting was performed by applying a flow of 300 µL/min buffer A ( 0 . 23% [v/v] formic acid [FA] ) to an ACQUITY UPLC BEH C18 1 . 7-μm , 2 . 1 × 5 mm Vanguard Pre-Column by an Agilent 1260 Infinity Quaternary pump ( Agilent Technologies , Santa Clara , CA ) . Peptides were separated on a 1 . 0 × 100 mm ACQUITY UPLC BEH C18 1 . 7-μm analytical column by a 12-min gradient from 95% buffer A to 50% buffer B ( 0 . 23% [v/v] FA in acetonitrile ) at a flow of 40 µl/min . Proteins were digested online with an Upchurch guard column ( 1 . 0 × 20 mm , IDEX , Oak Harbor , WA ) packed with agarose-immobilized pepsin ( Thermo Scientific Pierce , Rockford , IL ) . Peptides from peptic digests were identified from DDA MS/MS runs using ProteinLynx Global Server v2 . 4 ( Waters ) and MassAI v1 . 07 ( MassAI Bioinformatics , Stenstrup , DK , http://www . massai . dk ) . Deuterium incorporation for intact proteins and peptides was quantified with DynamX V2 . 0 ( Waters ) . Human GPIHBP1 ( UniProt id: Q8VI16 ) without the N- and C-terminal signal sequences was used for a homology search with the program HHPred ( Soding , 2005 ) . To find suitable reference structures , the PDB database was used to generate a multiple sequence alignment with human GPIHBP1; the top five highest-ranking proteins ( PDB entries: 20l3 , 2h7z , 2h5f , 3neq , 1hcp ) were selected as templates for homology modeling with MODELLER ( Sali et al . , 1995 ) . Molecular structures are displayed by PyMOL ( Schrödinger , LLC ) . A search for regions in human GPIHBP1 with a high propensity for intrinsic disorder was performed with the disorder prediction tools IUPred ( Dosztanyi et al . , 2005 ) and DISOPRED3 ( Jones and Cozzetto , 2015 ) . To initiate covalent cross-linking , mixtures of 1 . 5 µM LPL2 with either 7 µM GPIHBP11–131 , 7 µM GPIHBP134–131 , or 15 µM GPIHBP11–33 were exposed to 10 mM EDC for 90 min at 25°C in 10 mM Na2HPO4 ( pH 8 . 0 ) , 150 mM NaCl , 0 . 1% ( v/v ) Triton X-100 , and 10% ( v/v ) glycerol . The cross-linking reaction was terminated by boiling in SDS-PAGE sample buffer; samples were subsequently reduced and alkylated before analysis by SDS-PAGE to assess the formation of covalent complexes . Cross-linked tryptic peptides were identified by high-resolution mass spectrometry with a Q-Exactive HF mass spectrometer ( Thermo Scientific ) . Coomassie Blue–stained protein bands corresponding to cross-linked protein complexes were excised from the polyacrylamide gel and subjected to in-gel reduction , alkylation and digestion with trypsin , as described with minor modifications ( Shevchenko et al . , 1996 ) . Gel pieces were cut into smaller pieces , washed twice in 50% ethanol ( v/v ) and shrunk with 96% ( v/v ) ethanol . The shrunken gel pieces were then swollen in a 10 mM dithiothreitol ( DTT ) 0 . 1 M NH4HCO3 solution and incubated for 45 min at 56°C . After reduction , the solution was cooled to room temperature . The excess DTT solution was removed and replaced with a 55 mM iodoacetamide ( IAA ) 0 . 1 M NH4HCO3 solution followed by incubation for 30 min in the dark at room temperature . Excess IAA solution was removed; the gel pieces were washed twice in 50% ethanol ( v/v ) and the gel pieces were shrunken again using 96% ( v/v ) ethanol . Finally , the gel pieces were rehydrated on ice with a 100 mM NH4HCO3 solution containing 12 . 5 ng/µl trypsin . After 45 min , excess trypsin solution was removed and replaced with 100 mM NH4HCO3 , and the gel pieces were then incubated at 37°C overnight . The supernatant containing tryptic peptides was recovered the next day . Remaining peptides in the gel were extracted by adding 5% ( v/v ) formic acid , incubating for 15 min , and then adding an equal volume of acetonitrile and incubating for an additional 15 min . This supernatant was combined with the original supernatant and the entire pool dried in a vacuum centrifuge . Prior to analysis , the tryptic digest was resuspended in 0 . 1% trifluoroacetic acid ( TFA ) and desalted using solid-phase extraction in a pipet tip packed with Oligo R3 reversed phase resin ( Applied Biosystems ) . The column was subsequently washed with 0 . 1% ( v/v ) TFA; the bound sample was eluted with 0 . 1% ( v/v ) TFA in 70% ( v/v ) acetonitrile and then dried in a vacuum centrifuge . The samples were resuspended in 0 . 1% ( v/v ) FA and loaded onto an EASY-nLC system ( Thermo Scientific ) with a two-column setup . Trapping was performed with a 100 µm × 2 cm Acclaim PepMap100 C18 Nano-Trap Column and peptides were separated along a 75 µm × 25 cm Acclaim PepMap RSLC analytical column ( both Thermo Scientific ) . The peptides were eluted with an organic solvent gradient from 95% phase A ( 0 . 1% [v/v] FA ) to 25% phase B ( 0 . 1% [v/v] FA , 95% [v/v] acetonitrile ) over 80 min , followed by a 10-min gradient to 50% phase B at a flow rate of 250 nl/min . The eluted peptides were analyzed with a Q-Exactive HF mass spectrometer ( Thermo Scientific ) , operated in the positive ionization mode with data-dependent acquisition . Each MS scan was acquired at a resolution of 60 , 000 FWHM followed by 20 high-resolution HCD-MS/MS scans of the most intense ions . Ions selected for MS/MS were dynamically excluded for durations of 10 sec . MGF files were generated from the raw data with Proteome Discoverer v1 . 3 ( Thermo Scientific ) , and the protein crosslinks identified using MassAI ( Peng et al . , 2014 ) ( www . MassAI . dk ) . Searches against a database containing bovine LPL and GPIHBP1 R38G were performed with the following parameters: Precursor mass tolerance 10 ppm , MS/MS mass tolerance of 0 . 05 Da , tryptic cleavage allowing up to two missed cleavage sites , and carbamido-methylation of cysteine as a fixed modification . The following variable modifications were allowed: oxidation of Met; phosphorylation of Ser , Thr , and Tyr; and N-glycosylation of Asn-X-Ser/Thr/Cys . Peptide cross-links between Lys and Asp/Glu , with a resulting loss of water , were allowed . Cross-links were considered to be true if they had a score above 20 and the MS/MS spectra either contained fragment ion series originating from both peptides or contained peaks that represented cross-linked fragment ions .
GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase ( LPL ) within the interstitial space and shuttles it to the capillary lumen . The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing . The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry , surface plasmon resonance , and zero-length cross-linking . According to this model , GPIHBP1 comprises two functionally distinct domains: ( 1 ) an intrinsically disordered acidic N-terminal domain; and ( 2 ) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol . We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding . Importantly , the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain . This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia .
Fat is an important part of our diet . The intestines absorb fats and package them into particles called lipoproteins . After reaching the bloodstream , the fat molecules ( lipids ) in the lipoproteins are broken down by an enzyme called lipoprotein lipase ( LPL ) , which is located along the surface of small blood vessels . This releases nutrients that can be used by vital tissues – mainly the heart , skeletal muscle , and adipose tissues . LPL is produced by muscle and adipose tissue , but it is quickly swept up by a protein called GPIHBP1 and shuttled to its site of action inside the blood vessels . Mutations that alter the structure of LPL or GPIHBP1 can prevent the breakdown of lipids , resulting in high levels of lipids in the blood . This can lead to inflammation in the pancreas and also increases the risk of heart attacks and strokes . Many earlier studies have examined the properties of LPL , but our understanding of GPIHBP1 has been limited , mainly because it has been difficult to purify GPIHBP1 for analysis . Using genetically altered insect cells , Mysling et al . were able to purify two different forms of GPIHBP1 – a full-length version and a shorter version that lacked a small section at the end of the molecule known as the acidic domain . This revealed that the opposite end of the molecule – called the carboxyl-terminal domain – is primarily responsible for binding LPL and anchoring it inside blood vessels . Once LPL is bound to GPIHBP1 , the acidic domain of GPIHBP1 helps to stabilize LPL . If GPIHBP1’s acidic domain is missing then LPL is more susceptible to losing its structure , rendering it incapable of breaking down the lipids in the blood . Mysling et al . describe a new model for how LPL and GPIHBP1 interact that explains how specific mutations in the genes that encode these proteins interfere with the delivery of LPL to small blood vessels . In the future , this could help researchers to develop new strategies to treat people with high levels of lipids in their blood .