the binding of igg 
 fc to cell surface and soluble serum ligands 
 triggers a variety of immunological processes including phagocytosis , 
 cytotoxicity , inflammation , and immunosuppression .
the 
 structure of the fc domain is stabilized by the n - linked glycan , attached 
 to asn297 of each igg heavy chain .
this glycan is critical for fc function : 
 genetic or enzymatic removal leads to an almost complete loss of antibody 
 effector functions .
moreover , composition of the glycan 
 is a key parameter in determining the balance between pro - inflammatory 
 or anti - inflammatory effects .
for example , removal of the core 1,6-linked 
 fucose residue of the igg1 fc glycan enhances binding for fcriiia , while elevation of the levels of terminal 2,6-sialylation 
 leads to decreased natural killer cell activation and other potent cell - mediated immunosuppressive effects .
in addition to natural 
 variations in fc glycosylation , a growing number of enriched and engineered 
 fc glycoforms are finding application in therapeutic monoclonal antibodies 
 when a particular balance of effector functions is desirable .
the three - dimensional structure of the fc glycoforms has been investigated as have the independent effects of glycan and protein engineering 
 on receptor binding . by comparison
, however , relatively little is known about the interdependence 
 of glycan composition and protein structure on receptor binding .
the n - linked glycans in igg1 fc are complex , mostly core - fucosylated , 
 biantennary - type structures with varying amounts of bisecting glcnac , 
 terminal galactose , and sialic acid residues .
levels of sialylation are low with < 10% of total fc glycans from 
 serum igg being sialylated .
tri- or tetra - antennary 
 glycans are generally not found in serum igg fc .
the absence of larger , 
 branched , and/or sialylated structures is notable , especially when 
 compared to the glycosylation of other serum or cell - surface glycoproteins .
x - ray crystallographic and nmr studies of the igg fc domain have defined the conformation of the n - linked 
 glycans at asn297 . in the complex - type igg fc glycoforms ,
the conformation 
 of the oligosaccharide is well conserved and contacts over 500  of the surface of each c2 domains .
the 
 six arms of the glycan chain makes several stable interactions with 
 hydrophobic amino acid residues of the c2 domain .
the terminal 
 gal6 ( see legend to figure 1 for terminology ) on the six arm has been shown to restrict glycan 
 flexibility through interaction with the fc protein backbone .
key amino acid residues that interact with the 
 6-arm glycans through hydrogen bonds and hydrophobic interactions 
 include : phe241 , phe243 , val262 , val264 , asp265 , lys246 , and arg301 . 

aromatic rings of phe241 and phe243 form ch interactions 
 with the glcnac2 and glcnac5 residues 
 of the fc glycan and contribute to the stability of the fc domain .
the three arm on the other hand makes fewer contacts with the protein 
 backbone with hydrophobic interactions between man4 and 
 lys334 being the only observable protein  glycan interaction .
while the presence of extensive protein  glycan interactions 
 suggests a relatively immobile carbohydrate conformation and reduced 
 enzymatic processing , recent nmr spectroscopic studies indicate a 
 more dynamic and mobile role for the fc glycans .
nonetheless , the relatively limited processing of the fc glycan 
 indicates a reduced accessibility to glycan reactive enzymes in the 
 golgi apparatus .
mass spectrometric analysis of n - glycans released from 
 igg fc - f241a . 

( b ) the data from panel a were processed 
 with the maximum entropy 3 function of masslynx to convert multiply 
 charged ions to singly charged ions . the position of the fucose residue 
 in the triantennary glycans was not determined .
the ion at m / z 3169 gave a composition corresponding 
 to the tetrasialylated triantennary glycan , but this was not confirmed 
 by fragmentation .
( c ) an example of negative ion collision - induced 
 dissociation spectrum of the monosialylated , fucosylated biantennary 
 glycan .
( d ) spectra showing 
 trigalactosylated structures with three ( triply charged ) and four 
 ( quadruply charged ) sialic acids attached , respectively .
residue labeling follows that of vliegenhart 
 et al . with the additional modifications 
 of 7 for sialic acid , 1 for 16-linked 
 core fucose .
the symbolic representation 
 of glycans follows that of harvey et al . with residues in both the schematic diagrams and molecular graphics 
 following the color scheme of the consortium for functional glycomics .
the influence of hydrophobic residues 
 in the protein  glycan 
 interface of the fc on glycan processing was first observed in mutational 
 studies on mouse  human chimeric igg3 antibodies , where replacement 
 of phe241 , phe243 or val264 with ala resulted in elevated levels of 
 mono- and disialylated glycans and decreased binding to c1q and fcr 
 activity .
in addition , ribosomal display 
 has been used to discover site - specific fc mutants with enhanced fcriiia 
 binding .
this approach identified the 
 f243l mutant which , in addition to enhanced fcriiia binding , 
 also exhibited altered glycan processing including decreased fucosylation .
two possible explanations have been proposed 
 to explain these observations : either 
 the alteration of the protein  carbohydrate interaction directly 
 affected the fc protein conformation or the increased glycan processing 
 affected fcr binding . to discriminate between these possibilities , 

these glycoform - controlled mutants still 
 exhibited similar reduced 
 fcr binding indicating that the conformation of the c2 
 domain is modulated by glycan 
furthermore we show that the effects of these glycan - destabilizing 
 mutations on fcr binding are independent of mutations which 
 directly affect the fc  fcr interface .
thus , combinatorial 
 mutagenesis can yield antibodies with novel effector properties . for 
 example , we combine fc  fcr interface mutations known 
 to selectively enhance fcriib binding , with a glycan 
protein 
 interface mutation that decreases binding to all fcrs , to provide 
 an igg which more broadly eliminates activatory ( but not inhibitory ) 
 receptor binding .
this study therefore clarifies the role of the fc 
 glycan in maintaining antibody structure and provides routes to the 
 development of antibody therapeutics with bespoke effector functions .
glycan 
 interface , we determined the crystal structure of recombinant igg1 
 fc f241a ( table 1 ) .
this mutant has been previously 
 reported by lund et al . to reduce fcr binding and increase 
 glycan processing .
prior to crystallization , 
 we performed electrospray ionization ( esi ) mass spectrometric analysis 
 of the n - linked glycans which confirmed extensive branching and terminal 
 sialylation ( figure 1 ) .
the igg1 fc f241a 
 crystallized in the primitive orthorhombic spacegroup , p212121 , with one molecule of the 
 fc homodimer in the asymmetric unit .
data were collected to a resolution 
 of 1.9  ( table 1 ) . as observed in many 
 crystal structures of the fc ,
the protein and glycan moieties of the c2 
 domain from one chain ( chain b ) exhibited a high degree of disorder 
 ( figure 2d ) .
i(hkl)|/hkl ii(hkl;i ) , where i(hkl;i ) is the intensity of an individual 
 measurement and i(hkl 
 is the average intensity from multiple observations .
k|fcalc||/hkl |fobs| rfree is calculated as for rwork but using 
 only 5% of the data , which were sequestered prior to refinement .
interpretable electron density was observable for 
 seven monosaccharide 
 residues on chain a ( figure 2e , f ) . in the structure 
 of the wild - type fc , phe241 packs with the glcnac2 and 
 man3 residues ( figure 2b , c ) .
in contrast , electron density in our structure of the 
 f241a mutant , revealed the f241a site - directed substitution and a 
 destabilization of the protein  carbohydrate interface ( figure 2e , f ) .
the n - linked glycan rests in the established 
 position along the c2 domain with defined electron density 
 corresponding to the 6-arm , while only diffuse density corresponding 
 to the 3-arm is observed in the 2fo  fc map .
packing of n - link glycans in native ( a  c ) 
 and f241a mutant 
 ( d  f ) igg fc .
glycans are displayed as blue ( glcnac ) , red ( fuc ) , 
 and green ( man ) sticks .
protein is displayed as a gray cartoon with 
 four hydrophobic residues at the protein 
. overall structure of ( a ) native ( pdb i d 3ave ) and ( b ) f241a mutant 
 igg1 fc .
the c2 domains from chain a ( b and e ) are shown with 
 close - ups of the protein - glycan interfaces ( c and f ) .
electron density corresponding to carbohydrate is 
 depicted as a blue mesh ( 2fo  fc map contoured at 1 ) around the carbohydrate 
 moiety of the mutant fc reported herein .
this weak density indicates that 
 the 3-arm is either conformationally 
 disordered or glycoforms differing in 3-arm composition are adopting 
 different conformations where the x - ray scattering does not sum to 
 yield consensus electron density .
previous reports had suggested that 
 the intrinsic flexibility of the 3-arm causes the lack of electron 
 density of the galactose in native galactosylated fc .
consistent with a loss of electron density of the 3-arm , detailed 
 negative - ion esi mass spectrometry with fragmentation analysis revealed 
 extensive branching on the 3-arm but not the 6-arm ( figure 1 ) .
the localized induction of disorder at 
 the 3-arm does not fully 
 account for the apparent increase in accessibility of the glycans 
 to golgi - resident glycosyltransferases ( e.g. , increase in 6-arm galactosylation ) 
 or the reduction in fcr binding affinity ( figure 2 ) .
protein interface not sufficiently 
 captured by low - temperature x - ray crystallographic methods . for example , the hydrophobic interface mutations 
 may affect the position of equilibrium between the protein 
bound 
 and  free conformations proposed by nmr studies to 
 yield more accessible glycans and potentially more widely spaced c2 
 domains .
interestingly , nmr studies have 
 indicated that 2,6-sialylation has minimal impact on glycan 
 dynamics in the fc domain .
this finding 
 is entirely consistent with our observation of an elevation in 6-arm 
 sialylation by electrospray mass spectrometry and the conserved conformation 
 of the 6-arm observed by x - ray crystallography ( figure 2c , f ) .
protein 
 interface can induce at least localized glycan disorder , we next sought 
 to determine to what extent the reduction in fcriiia binding 
 can be attributed to disruption of the glycan  protein interface 
 or to changes in glycan processing . to this end
a series of human 
 igg1 fc mutants was generated containing mutations that modulated 
 the protein  carbohydrate interactions within the fc : f241a , 
 f243a , v262e , and v264e .
the panel of igg1 b12 mutants was expressed 
 in either human embryonic kidney ( hek ) 293 t or glcnac transferase 
 ( gnt ) i - deficient hek 293s cells .
the 
 glycans were released from the purified antibody via protein n - glycosidase 
 f ( pngase f ) .
the free sugars were fluorescently labeled and resolved 
 via normal - phase high - performance liquid chromatography ( hplc ) using 
 a tsk - amide column .
the hplc spectra from igg1 fc mutants expressed 
 in hek 293 t ( black spectra ) or gnt i - deficient hek 293s cells ( blue 
 spectra ) are shown in figure 3 .
the glycans 
 from igg b12 expressed in hek 293 t cells are composed of a series 
 of fucosylated , biantennary , complex - type carbohydrates , typical of 
 the protein - directed glycosylation observed for igg ( figure 3a ; black spectrum ) .
the most abundant species observed 
 were agalactosylated structures with smaller amounts of mono and digalactosylated 
 structures .
a small population of sialylated material was also present , 
 showing the typical glycan profile for recombinant igg1 fc as reported 
 previously .
consistent with previous analyses 
 of igg3 , mutations interrupting the hydrophobic 
 protein  glycan interface led to dramatic increases in terminal 
 1,4 linked galactose levels , with digalactosylated species 
 representing the most abundant glycan populations for all mutants 
 generated from hek 293 t cells ( figure 3b  e ; 
 black spectra ) .
tri- and tetra - antennary species , not normally observed 
 on fc , were detected , most notably on v262e and v264e . additionally , 
 increased bisecting glcnac and terminal sialylation were also evident 
 for these mutants .
an unusual digalactosylated , trisialylated species 
 was also detected in the hplc spectra of all of the mutants .
this structural assignment was 
 confirmed by electrospray mass spectrometry of recombinant igg1 fc 
 ( figure 1 ) and b12 mutants ( figures s1 and s2 ) .
further matrix - assisted laser desorption / ionization 
 time - of - flight mass spectrometry ( maldi - tof - ms ) ( figure s3 ) and hplc analysis of desialylated mutants ( figures s4 and s5 ) were carried out to validate 
 the hplc assignments .
in contrast to the heterogeneous complex - type 
 glycan spectra observed 
 when the igg panel was expressed in hek 293 t cells , expression in 
 gnt i - deficient hek 293s cells led to a purely oligomannose - type glycan 
 profile composed of man5glcnac2 together with 
 a small population of fucosylated man5glcnac2 ( figure 3 ; blue spectra ) . the latter structure 
 has previously been shown to arise via the inefficient gnt i - independent 
 fucosylation pathway .
protein interface affected neither 
 oligomannose processing to man5glcnac2 nor the 
 proportion of gnt i - independent fucosylation .
in addition to glycan 
 analysis , the n - glycosylation site occupancy at the asn297 was examined , 
 as previous report showed decreased glycan site occupancy for the 
 hydrophobic mutants .
a combination of 
 pngase f treatment and trypsin digestion , followed by maldi - tof - ms , 
 showed that the n - glycosylation site on fc asn297 is fully occupied 
 for all the mutants ( figures s6 and s7 ) . 

the discrepancy might be due to the different antibody isotypes used 
 or methods used to examine site occupancy .
hplc analysis of 2aa - labeled 
 n - linked glycans from monoclonal igg1 
 b12 mutants expressed in hek 293 t and hek 293s cells .
normal - phase 
 hplc analysis of 2-aa - labeled n - linked glycans released from target 
 antibody glycoforms by in - gel protein pngase f digestion .
glycan profile 
 of igg1 b12 expressed in hek 293 t ( black ) and hek 293s ( blue ) for 
 the following variants : ( a ) wild type ; ( b ) f241a ; ( c ) f243a ; ( d ) v262e ; 
 and ( e ) v264e .
the y - axis displays relative fluorescence 
 ( rf ) . to investigate the impact of sialylation 
 on fcr binding , in vitro glycosyltransferase 
 reactions were used to generate 
 hyper-2,6-sialylated igg ( figure 4 ) . 
 as there is a potential for 2,3-linked sialic acid to be present 
 in glycoproteins derived from hek 293 t cells , we first performed a 
 sialidase digestion .
sialylation was not detected by hplc analysis 
 ( figure 4a , b ) . the sialidase - treated material 
 was then subjected to sequential 1,4-galactosylation and 2,6-sialylation 
 ( figure 4c , d , respectively ) .
hplc analysis 
 revealed that a trace population ( 5% ) of monogalctosylated 
 glycans remained after the galactosyltransferase reaction ( figure 4c ) .
generation of differentially glycosylated igg1 fc . normal - phase 
 hplc analysis of 2-aa - labeled n - linked glycans , released from target 
 antibody glycoforms by in - gel pngase f digestion . ( a ) glycan profile 
 of monoclonal igg1 b12 .
( b ) glycan profile of igg1 incubated with 
 50 u / ml clostridium perfringens neuraminidase 
 for 48 h at 37 c .
( c ) glycan profile of igg1 incubated with 
 25 g / ml 1,4-galactosyltransferase ( b4galti ) and 80 m 
 uridine 5-diphosphogalactose in 50 mm hepes , 10 mm mncl2 , ph 7.5 for 48 h at 37 c .
( d ) glycan profile of igg1 
 sequentially treated with b4galti and 2,6-sialyltransferase 
 i ( st6gali ) as described above .
 fcrs by determining the binding of 
 our panel of igg mutants to the extracellular regions of fcria , 
 fcriia , fcriib , fcriiia , and fcriiib ( figure 5 ) .
elisa of monoclonal igg variants binding human fcria , 
 fcriia , 
 fcriib , fcriiia , and fcriiib .
the fcrs 
 were plated at 5 g / ml overnight at 4 c , igg variants 
 f241a , f243a , v262e , and v264e were incubated for 1.5 h , and binding 
 was detected by hrp - conjugated goat antihuman fab antibody
. symbolic 
 representation of igg mutation and glycovariants : solid black square 
 = wild - type native ; solid blue square = wild - type man5glcnac2 ; open black triangle = mutant native ; open blue triangle 
 = mutant man5glcnac2 ; solid red square = wild - type 
 hypergalactosylated and hypersialylated , elisa binding curves of the 
 four igg hydrophobic mutants for ( a ) fcria , igg variant starting 
 concentration at 10 g / ml . ( b ) fcriia , igg variant starting 
 concentration at 100 g / ml . ( c ) fcriib , igg variant starting 
 concentration at 300 g / ml . ( d ) fcriiia , igg variant 
 starting concentration at 100 g / ml . ( e ) fcriiib , igg 
 variant starting concentration at 300 g / ml . ( f ) fcriiia , 
 igg variant starting concentration at 100 g / ml . all data points 
 represent the calculated mean of two independent measurements from 
 a total of at least two experiments .
consistent with studies with fcri , our data show that 
 abolition 
 of the fc glycan  protein hydrophobic interaction resulted in 
 a decreased fc affinity for the fcria ( figure 5d ) . a similar pattern , albeit 
 to different extents
, was observed in the binding analysis with fcriiia , 
 fcriia , fcriib , and fcriiib ( figure 5 ) . the high affinity , activatory fcria and 
 low - affinity , inhibitory fcriib were least prone to such modulation . 

interestingly , the v264e mutant contains the highest level of sialylated 
 structures among mutants tested ( figure 3e ) . 

. the v264e mutation would be 
 predicted to perturb the overall trajectory of the glycan away from 
 the surface of the c2 domain and may account for the extensive 
 glycan terminal processing due to increased steric accessibility . to investigate the effect of these hydrophobic mutations on fcr 
 binding independent of the differential fc glycoforms , we compared 
 fcr binding to the panel of uniformly glycosylated igg1 fc 
 mutants expressed in the gnt i - deficient hek 293s cells ( figure 3 ; blue spectra ) .
consistent with previous 
 reports , wild - type igg1 fc with man5glcnac2 glycosylation 
 exhibits increased affinity 
 for fcriiia ( figure 5d ) and decreased 
 affinity for the fcriib ( figure 5e ) 
 compared with fc with native biantennary complex glycans .
we also demonstrate that the man5glcnac2 glycoform 
 exhibits decreased fc affinity for fcriia , which is highly 
 homologous to fcriib ( figure 5b , c ) .
mutations that disrupt the hydrophobic interface significantly 
 decrease the fc affinity for fcriia , fcriiia , and fcriiib , 
 largely independently of the glycoform .
this effect is evident by 
 comparison of the wild - type and mutants expressed in gnti - deficient 
 hek 293s cells ( figure 3 ; blue spectra ) .
mutations 
 affecting the hydrophobic interface resulted in significantly decreased 
 fc affinity for fcriia , fcriiia , and fcriiib 
 ( figure 5b , d , e ) , while interestingly , no significant 
 changes were observed for fcriib binding ( figure 5c ) . for the high affinity fcria ,
together , 
 our results show that the hydrophobic mutations disrupt the fc binding 
 to the activatory fcrs independently of the fc glycan , indicating 
 that the productive engagement of fc  fcr requires the 
 protein  glycan interaction at the fc c2 domain . unlike f241a , v262e , and v264e ,
the f243a mutant expressed as the 
 man5glcnac2 glycoform has a minimal effect on 
 fcrs binding ( figure 5 ) .
this minimal 
 effect can be explained by the different protein  glycan interfaces 
 of oligomannose- , hybrid- , and complex - type antibody glycoforms as 
 revealed by x - ray crystallography . in 
 contrast to the other residues , f243 exhibits minimal van der waals 
 contacts with the 6-arm mannose residues in the predicted structure 
 of the man5glcnac2 glycoform .
therefore , in contrast to the significant effect of f243a 
 mutation on the mobility of complex - type glycans , f243a would be predicted 
 to have a minimal impact on the mobility of man5glcnac2 structures .
the relative affinity of the single mutants for 
 fcrs is summarized in table s2 .
remarkably , the interaction between fcriib and igg1 fc is 
 relatively unperturbed by the disruption of fc protein 
we hypothesized that 
 further enhancements to the fcriib selectivity could be achieved 
 by combining our glycan 
protein interface mutations with previously 
 reported hinge - proximal c2 mutations ( s267e / l328f ) that exhibit 
 selective fcriib - binding .
we generated 
 two novel igg1 fc mutants , v262e / s267e / l328f and v264e / s267e / l328f , 
 which exhibited enhanced affinity for the fcriib and , by comparison 
 to the double mutant alone , significantly decreased 
 affinity for fcria , fcriia , and fcriiib ( figure 6 ; table s3 ) .
the double 
 mutant s267e / l328f shows significantly increased affinity for the 
 inhibitory fcriib and decreased affinity for the activatory 
 fcriiia , while the binding for the other fcrs remains 
 similar to the wild type ( figure 6 ) , consistent 
 with published data . the increased levels 
 of glycan terminal processing and bisection of these triple mutants 
 are comparable to those of the single v262e and v264e mutants ( figure s8 ) .
elisa of monoclonal igg variants binding 
 human fcria , fcriia , 
 fcriib , fcriiia and fcriiib .
the fcrs 
 were plated at 5 g / ml overnight at 4 c , igg variants 
 s267e / l328f , v262e / s267e / l328f , and v264e / s267e / l328f were incubated 
 for 1.5 h and binding was detected by hrp - conjugated goat antihuman 
 fab antibody .
symbolic representation of igg mutation and glycovariants : 
 solid black square = wild - type native ; solid blue square = wild - type 
 man5glcnac2 ; open black triangle = mutant native ; 
 open blue triangle = mutant man5glcnac2 , elisa 
 binding curves of the four igg hydrophobic mutants for ( a ) fcria , 
 igg variant starting concentration at 10 g / ml . ( b ) fcriia , 
 igg variant starting concentration at 100 g / ml . ( c ) fcriib , 
 igg variant starting concentration at 300 g / ml . ( d ) fcriiia , 
 igg variant starting concentration at 100 g / ml . ( e ) fcriiib , 
 igg variant starting concentration at 300 g / ml . all data points 
 represent the calculated mean of two independent measurements from 
 a total of at least two experiments .
protein 
 interactions modify the fc structure relevant for fcr binding 
 ( figure 5 ) .
however , it has previously been 
 shown that hypersialylation of igg1 fc , regardless of the linkage 
 type , decreases fc binding to the fcria , fcriib , and 
 fcriiia , which also reduces antibody - mediated cytotoxicity 
 both in vivo and in vitro . therefore , we generated hypergalactosylated 
 and hypersialylated human igg1 fc ( figure 4 ) and examined their affinity for fcriiia , a critical determinant 
 of natural killer cell - mediated adcc .
usually , the fc  fcr 
 affinity strongly correlates with effector functions measured by cellular 
 assays .
however , our data indicate that the hypersialylated 
 fc binds to the fcriiia with very similar affinity to the wild 
 type , supported by both elisa ( figure 5f ) and 
 surface plasmon resonance ( spr ) data ( figure 7 ) .
one possible explanation for the reduced cytotoxicity is that 
 terminal sialic acid might interact with inhibitory sialic acid binding 
 ig - like lectins ( siglec ) present on the surface of immune cells including 
 macrophages and natural killer cells .
this minimal effect of fc sialylation on fcriiia binding 
 is also supported by the structural analysis presented here , which 
 reveals that the protein 
glycan interface , rather than glycan 
 terminal processing , modulates c2 domain conformation ( figure 5 ) .
the igg variants were injected at 5 different concentrations 
 at a flow rate of 30 l / min : igg and igg hypersialylated ( 0.67 , 
 0.33 , 0.17 , 0.083 , and 0.042 m ) ; igg man5glcnac2 ( 0.33 , 0.17 , 0.083 , 0.042 , and 0.021 m ) .
sensorgrams were fitted with a global 
 1:1 interaction , and the ka , kd , and kd were calculated , 
 all using bia evaluation software 2.0.3 .
kd values are reported as mean  sd , and sensorgrams are representative 
 a total of three independent experiments .
modulation of the multiple hydrophobic 
 interactions between the 
 protein surface and the glycan within the igg fc strongly influences 
 fcr binding , presumably by altering the dynamics of the fc 
 and affecting the adoption of receptor binding conformations .
while 
 these mutations also modulate glycan processing , expression of fc 
 domains with homogenized glycoforms revealed that the interaction 
 between the glycan and the protein independently influences fcr 
 binding . by combining interface mutations with glycan engineering , 
 new portfolios of effector activities can be generated .
the pfuse vector 
 with the human igg1 fc insert was obtained from invivogen , u.k .
the 
 vectors encoding igg1 b12 light and heavy chains were kindly provided 
 by prof .
protein mutagenesis was performed using the 
 quikchange kit ( agilent technology , u.k . ) to generate the igg1 fc 
 mutant f241a and the full - length igg1 b12 mutants f241a , f243a , v262e , 
 and v264e .
the mutated fc ( residues 225447 , swiss - prot accession 
 number p01857.1 ) encompassing hinge , c2 and c3 domains was cloned 
 into the mammalian expression vector , phlsec .
the vectors containing 
 full - length fcria , fcriia ( his131 variant ) , fcriib , 
 fcriiia ( val158 variant ) , fcriiib , and mouse -1,4-galactosyltransferase 
 i ( b4galti ) were all purchased from open biosystems , u.k .
the vector 
 containing full - length rat 2,6-sialyltransferase i ( st6gali ) 
 was a gift from prof .
the soluble extracellular regions of each fcr , b4galti and 
 st6gali were cloned into the phlsec vector as described for the fc : 
 fcria ( residues 16288 ; swiss - prot accession number bc152383 ) ; fcriia 
 ( residues 34217 ; swiss - prot accession number bc020823 ) ; fcriib 
 ( residues 42225 ; swiss - prot accession number nm_001190828 ) fcriiia ( residues 16288 ; swiss - prot accession number bc033678 ) ; fcriiib 
 ( residues 17200 ; swiss - prot accession number bc128562 ) ; st6gali 
 ( residues 89403 ; swiss - prot accession number np_001106815 ) ; and b4galti ( residues 127399 ; swiss - prot accession number bc053006 ) . for 
 b4galti , the residue cys339 was mutated to thr to minimize the potential 
 for aggregation .
the fc , fcrs , b4galti , and 
 st6gali were expressed in hek 293 t cells as previously described .
briefly , hek 293 t cells ( atcc number crl-1573 ) 
 were grown to 90% confluence and transiently transfected with polyethyleneimine 
 ( pei ) , using a transfection mix with 
 dna and pei in ratio of 1:1.5 . following transfection ,
cells were 
 grown in dmem/1% fetal bovine serum at 37 c and 5% co2 for 5 days .
protein was purified from cell supernatant by immobilized 
 metal affinity chromatography using chelating sepharose fast flow 
 ni - agarose beads ( ge healthcare , u.k . )
followed by size 
 exclusion chromatography using a superdex s-200 column equilibrated 
 in phosphate buffered saline ( pbs ) ( for fcrs ) or 10 mm hepes 
 ph 7.4 , 150 mm nacl ( for fc ) .
full - length igg1 b12 was transiently 
 expressed in hek 293 t or gnt i - deficient hek 293s cells .
prior to transfection , light and heavy chain plasmids were mixed 
 in a mass ratio of 4:1 , and the total dna was mixed with pei in a 
 mass ratio of 1:1.5 . after incubation for 4 days at 37 c , cell 
 culture supernatant was harvested and igg1 b12 was purified using 
 protein a sepharose ( ge healthcare , u.k . ) according to the manufacturer s 
 directions .
oligosaccharides 
 were released from coomassie blue - stained reducing sds - page gel bands 
 containing 40 g of igg fc as previously reported .
gel bands were rehydrated with 30 l of 
 30 mm nahco3 ph 7.0 containing 100 units / ml pngase f ( new 
 england biolabs , u.k . ) and incubated for 12 h at 37 c .
desialylation was carried out using linkage 
 nonspecific neuraminidase from clostridium perfringens ( new england biolabs , u.k . ) for 48 h at 37 c .
glycans were 
 labeled with anthranilic acid ( 2-aa ) as previously described and separated using tsk amide column ( sigma - aldrich , 
 u.k . ) .
the released n - glycans , dissolved in water and 2-aa labeling 
 buffer ( 3/8 , v / v ) were mixed with 2-aa and sodium cyanoborohydride 
 and incubated for 1 h at 80 c ; excess 2-aa dye was removed using 
 a spe - ed amide-2 column ( systematic systems , u.k . ) .
solvent 
 a was acetonitrile , solvent b was milli - q water , and solvent c was 
 800 mm ammonium hydroxide adjusted to ph 3.85 using acetic acid .
the gradient 
 was a constant 71.6% a for 6 min at a flow rate of 0.8 ml / min , followed 
 by a linear gradient of 71.635% a over 80 min at 0.8 ml / min . 

afterward , the gradient was a linear 3571.6% a for 1 min at 
 a flow rate of 0.8 ml / min ; then at the same gradient , the flow rate 
 increased from 0.8 ml / min to 1.2 ml / min over 1 min and followed by 
 the same gradient and flow rate for 13 min .
the run finished by returning 
 the flow rate to 0.8 ml / min over 1 min .
fluorescence was detected 
 at 425 nm , and the excitation wavelength was 360 nm .
chromatography 
 data were processed by the empower software ( all instruments and software 
 from interlink scientific services limited , u.k . ) .
assignments were 
 consistent with previously reported serum igg n - linked glycan profiles and were confirmed by maldi - tof ms and exoglycosidase 
 analysis .
hyper-2,6-sialylated igg was generated by incubating with 
 b4galti in the presence of 80 m uridine 5-diphosphogalactose 
 ( sigma - aldrich , u.k . ) in 50 mm hepes , 10 mm mncl2 , ph 7.5 
 for 48 h at 37 c .
the hyper-1,4-galactosylated igg was 
 treated with st6gali in the presence of 70 m cytidine-5-monophospho - n - acetylneuraminic acid ( sigma - aldrich , u.k . ) in 50 mm hepes , 
 10 mm mncl2 , ph 6.5 for 48 h at 37 c .
the composition 
 of the glycoform was verified by hplc analysis after each enzymatic 
 treatment .
recombinantly 
 expressed mutant igg1 fc ( f241a ) was concentrated to 7.0 mg / ml and 
 crystallized after 10 days using the sitting drop vapor diffusion 
 method using 100 nl protein plus 100 nl precipitant equilibrated against 
 95 l reservoirs .
crystals of f241a igg1 fc grew at room temperature 
 in a precipitant containing 28% polyethylene glycol monomethyl ether 
 2000 in 0.1 m bis - tris buffer at ph 6.5 .
crystals were flash frozen 
 by immersion in a cryoprotectant containing the mother liquor diluted 
 in 30% polyethylene glycol and then rapidly transferred to a gaseous 
 nitrogen stream .
crystallographic data were collected to 1.9  
 resolution at beamline i04 at the diamond light source ( oxfordshire , 
 u.k . ) .
the structure was solved using molecular replacement 
 with the program phaser using native 
 fc ( pdb accession no .
model building was performed with 
 coot and iteratively refined using restrained 
 refinement in the ccp4 supported program , refmac5 , with the incorporation 
 of translation - libration - screw ( tls ) parametrization and automatically 
 generated local noncrystallographic symmetry restraints.model quality was validated with molprobity .
recombinant fcrs at 
 5 g / ml in pbs were coated on high - binding microtiter plates 
 ( 3690 , corning , ny , u.s.a . ) overnight at 4 c .
coated plates 
 were washed with pbs containing 0.05% tween 20 ( sigma - aldrich , u.s.a . ) 
 and blocked for 1 h at room temperature with 5% bovine serum albumin 
 ( bsa ) in pbs .
recombinant igg1 b12 expressed from hek 293 t cells or 
 from gnt i - deficient hek 293s cells were then added and allowed to 
 bind for 1.5 h at room temperature .
plates were washed five times 
 with pbs containing 0.05% tween and binding was detected using a horse 
 radish peroxidase ( hrp)-conjugated fab fragment specific for human 
 igg fab ( abcam , u.k . ) .
the 3,3,5,5-tetramethylbenzidine 
 substrate ( tmb ; thermo scientific , u.s.a . ) was used for development 
 according to the manufacturer s directions and was stopped 
 by the addition of 2 m h2so4 .
absorbance was 
 measured at 450 nm on a spectramax m5 ( molecular devices , california , 
 u.s.a . )
apparent affinity was calculated as 
 the concentration of igg1 b12 corresponding to half - maximal binding 
 on the elisa binding curve .
spr experiments 
 were carried 
 out using the biacore t100 instrument ( ge healthcare , u.k . ) .
briefly , 
 the fcriiia ( val158 variant ) was immobilized onto the surface 
 of cm5 sensor chip ( ge healthcare , u.k . ) to about 1000 ru during each 
 independent experiment .
all experiments were carried out in the hbs - ep 
 running buffer ( 10 mm hepes , 150 mm nacl , 3 mm edta and 0.005% surfactant 
 p20 ) , at a flow rate of 30 l / min .
the monoclonal igg1 b12 protein 
 and the hypersialylated and high - mannose glycol variants were injected 
 at 5 different concentrations , allowing 2 min for association and 
 3 min for dissociation .
after each run , the sensor chip was regenerated 
 using 10 mm glycine - hcl , ph1.7 .
the sensorgrams were fitted to a global 
 1:1 interaction , and the ka , kd , and kd were calculated , 
 all using biaevaluation software 2.0.3 ( ge healthcare , u.k . ) .
 biologically active 
 conformations of the igg1 fc homodimer are 
 maintained by multiple hydrophobic interactions between the protein 
 surface and the n - glycan . 
 the fc glycan modulates biological effector 
 functions , including antibody - dependent cellular cytotoxicity ( adcc ) 
 which is mediated in part through the activatory fc receptor , fcriiia . 
 
 consistent with previous reports , we found that site - directed mutations 
 disrupting the protein  carbohydrate interface ( f241a , f243a , 
 v262e , and v264e ) increased galactosylation and sialylation of the 
 fc and , concomitantly , reduced the affinity for fcriiia . 
 we 
 rationalized this effect by crystallographic analysis of the igg1 
 fc f241a mutant , determined here to a resolution of 1.9  , which 
 revealed localized destabilization of this glycan  protein interface . 
 
 given that sialylation of fc glycans decreases adcc , one explanation 
 for the effect of these mutants on fcriiia binding is their 
 increased sialylation . 
 however , a glycan - engineered igg1 with hypergalactosylated 
 and hypersialylated glycans exhibited unchanged binding affinity to 
 fcriiia . 
 moreover , when we expressed these mutants as a chemically 
 uniform ( man5glcnac2 ) glycoform , the individual 
 effect of each mutation on fcriiia affinity was preserved . 
 
 this effect was broadly recapitulated for other fc receptors ( fcri , 
 fcriia , fcriib , and fcriiib ) . 
 these data indicate 
 that destabilization of the glycan  protein interactions , rather 
 than increased galactosylation and sialylation , modifies the fc conformation(s ) 
 relevant for fcr binding . 
 engineering of the protein  carbohydrate 
 interface thus provides an independent parameter in the engineering 
 of fc effector functions and a route to the synthesis of new classes 
 of fc domain with novel combinations of affinities for activatory 
 and inhibitory fc receptors . 
Introduction
Results
and Discussion
Conclusions
Experimental Procedures