Nucleotide sequence of cDNA encoding human a2-macroglobulin and assignment of the chromosomal locus (human serum proteins/proteinase inhibitors/human chromosome mapping/thiol ester bond)


Six a2-macroglobulin (a2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, pa2M1, carries a 4.6-kilobase-pair insert, which was se- quenced. The insert contains the coding sequences for the ma- ture a2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor pro- a2M. At the 3' end of the insert a poly(A) addition signal A-A- T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published a2M amino acid sequence for all except three residues. The a2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using a2M cDNA as a hybridization probe. 

a2-Macroglobulin (a2M) is a serum glycoprotein and a major plasma proteinase inhibitor with a wide specificity. a2M-related proteins are present in all vertebrate species (1)(2)(3)(4). Hu- man a2M is a tetramer of four identical 185-kDa subunits, arranged as a pair of dimers each consisting of two disulfide- linked monomers (5,6). The a2M polypeptide has a so-called bait region and an internal thiol ester bond, which account for its properties as a proteinase inhibitor. The bait region, composed of a series of target peptide bonds for plasma pro- teinases (7, 8), is located close to the center of the monomer- ic subunit. The thiol ester bond is a covalent linkage between the y-carboxyl function of glutamate residue 975 and the sul- hydryl group of cysteine residue 972 (9-12). Cleavage of the bait region by proteinases leads to a conformational change of the a2M molecule. This conformational change activates the thiol ester bond, leading to its hydrolysis and as a conse- quence to covalent binding between the proteinase and a2M via the reactive glutamate 975. a2M receptors are present on the surfaces of fibroblasts and macrophages. These receptor proteins preferentially bind a2M-proteinase complexes or a2M molecules with hydrolyzed thiol ester bonds (13, 14). This suggests that the conformational change, which accom- panies the complex formation between a2M and proteinases and the hydrolysis of the thiol ester bond, exposes regions of the a2M molecule that are recognized by these receptors. Receptor-mediated endocytosis of proteinase-a2M com- plexes by macrophages and liver cells leads to clearance of the complexes from the circulation. 
Internal thiol ester bonds are also found in the complement proteins C3 and C4 (15), which are derived from pre- cursor polypeptides of size similar to a2M (180-200 kDa). Their thiol ester sites are found in positions comparable to those in a2M, and the amino acid sequences of all three thiol ester sites are conserved. These observations led to the pro- posal that the C3, C4, and a2M genes are derived from a common ancestral gene (16). The sequences of murine and human C3 and human C4 and partial cDNA sequences of murine C4 have recently been determined (17-21). Compari- son of human a2M with murine C3 revealed a 25% overall sequence homology (17, 18, 22), supporting the proposal of their common evolutionary origin. The human C3 gene is lo- cated on chromosome 19 (23), and the human C4 genes map to the histocompatibility complex on chromosome 6 (24). 
In addition to binding to proteinases, a2M can also adhere to small molecules, including lectins, cations (zinc), basic proteins (myelin basic protein, histone H4), and platelet-de- rived growth factor (PDGF) (1, 25). The binding mechanisms involved are probably unrelated to the trapping of protein- ases because adherence of small molecules does not prevent the proteinase binding (1). A physiological role for the com- plex formation between a2M and these small molecules has not been established. The striking similarity of the amino acid sequences of PDGF and p28Sis, the transforming protein of the simian sarcoma virus (26,27), suggests that viral and other cellular transformation mechanisms may be mediated by growth factor-like proteins. Thus, a2M may serve as an important role in limiting the effects of PDGF-like mitogens. 
The amino acid sequence of a2M has recently been reported (28). For our future studies of the structural properties of a2M that underlie its functions and of the regulation of expression of the a2M gene, it is necessary to prepare cDNA clones and to derive the complete cDNA sequence. Here we report the isolation and sequence analysis of human a2M cDNA clones and the assignment of the human a2M locus to chromosome 12. 

MATERIALS AND METHODS

Materials. Restriction enzymes were from Boehringer Mannheim, DNA polymerase I (Klenow fragment) was from Bethesda Research Laboratories, and the 17-nucleotide uni- versal sequencing primer was from Collaborative Research. T4 DNA polymerase and dideoxyand deoxynucleotide tri- phosphates were from P-L Biochemicals. The radionucleo- tides [a-32P]dCTP (>400 Ci/mmol; 1 Ci = 37 GBq) and deoxyadenosine 5'-(a-[35S]thio)triphosphate (>400 Ci/ mmol) were from Amersham. T4 DNA ligase and the Esche- richia coli strain JM101-TG1 were gifts from D. Bentley and T. Gibson. Oligonucleotide probes were synthesized by Cre- ative Biomolecules (South San Francisco, CA). Synthetic Oligonucleotide Probes and Isolation of cDNA Clones. The construction of the human liver cDNA library enriched for mRNA species over 2000 nucleotides in length Abbreviations: C3 and C4, third and fourth components of complement; a2M, a2-macroglobulin; kb, kilobase pair(s). 
with the plasmid vector pAT 153 Pvu II-8 has been previous- ly described (19). Two methionine residues at positions 1378 and 1385 of the human a2M sequence (28) and their surrounding sequences were chosen as probe sites because they were encoded by nucleotide triplets with minimal degenera- (N represents all four nucleotides) and 
S5'-A-C-C-A-T-C _T-T-N-A-C-G T-C-3'.
This cDNA library consisted of about 15,000 different recombinant clones; 30,000 colonies were plated on nitrocel- lulose discs. A set of replica filters were prepared from the master filters (29). Colonies on replica filters were grown, then transferred to chloramphenicol plates to amplify the plasmid. The colonies were processed (30, 31) and hybridized with kinase 32P-labeled (32) 14-mer and 17-mer oligonucleotide mixtures. Positive colonies were rescreened until genetic homogeneity was reached. The length of cDNA in- serts was determined by restriction analysis with the en- zymes HindIII and Sal I. 
DNA Sequence Analysis. Plasmid DNAs carrying the longest cDNA inserts were digested with HindIII and Sal I. The fragments were separated by electrophoresis in low-melting agarose gels and recovered from the agarose by extraction with phenol. Shotgun cloning of random 200-to 400-basepair subfragments in the Sma I site of the phage M13 mp8 vector and preparation of single-stranded DNA from phage particles were performed as described (33). DNA sequences were determined with the dideoxynucleotide chain-termination method (34), using deoxyadenosine 5'-(a-[35S]thio)triphosphate and buffer gradient gels (35). DNA sequence data were analyzed by using Staden's computer programs (36- 38). 
Chromosomal Assignment of the a2M Locus. All human/ rodent somatic cell hybrids grown for DNA preparations (Table 1), have been recharacterized as to human chromo- some composition by karyotyping (39,40), human isozyme analysis (41,42), or both. High molecular weight DNA from human, rodent and hybrid clones was digested with the re- striction enzymes EcoRI, Pst I (Boehringer Mannheim), or Taq I (P-L Biochemicals). Resulting fragments were electrophoretically separated in 0.7% agarose gels. DNA was dena- tured and transferred to nitrocellulose filters (Schleicher & Schuell) (43). Filters were hyridized with nick-translated (44) a2M cDNA in 2.5x NaCl/Cit (lx NaCl/Cit is 150 mM sodi- um chloride/15 mM trisodium citrate), lOx Denhardt's solu- tion (2% Ficoll/2% bovine serum albumin/2% polyvinylpyrrolidone), 0.1% sodium dodecyl sulfate, 6% polyethylene glycol 6000 (Fisons, Loughborough, England) or 10% dex- tran sulfate, and denatured salmon sperm DNA at 50 ,ug/ml. Hybridization was carried out at 65°C overnight. Filters were washed at 65°C successively with 2x, 1x, 0.3x, and 0.lx NaCl/Cit, three times for 20 min each. 

RESULTS

Identification and Sequence Analysis of cDNA Clones. A human liver cDNA library was screened with two mixed synthetic oligodeoxynucleotide probes, a 17-mer and a 14- mer constructed on the basis of known amino acid se- quences. Of 30,000 colonies screened, 34 clones hybridized with both the 17-mer and 14-mer probes. 
After restriction enzyme analysis of the cDNA inserts, six recombinant plamids were selected for detailed analysis. One of them, pa2M1, carries a 4.6-kilobase pair (kb) insert whose nucleotide sequence was determined. The nucleotide sequence from the 5' nontranslated region to the poly(A) tail of the mRNA and the derived amino acid sequence are given in Fig. 1. The complete coding sequence was determined on both DNA strands; each character was determined 3.75 times on average. A methionine encoding triplet (nucleotides 44 to 46) was identified as the translational initiation site. This methionine, together with the following 22 amino acid residues, forms the signal peptide of the a2M protein, which has not previously been described. Thus, a2M must be syn- thesized as a pro-a2M precursor polypeptide. The a2M signal peptide has a 10 out of 23 amino acid residue sequence homology with the mouse complement C3 signal peptide (Fig. 2 and ref. 17). This similarity further supports the idea that the C3 and a2M genes must have a common evolution- ary origin (17,22). The amino acid sequence derived from the cDNA sequence was compared with the a2M amino acid sequence determined on the protein level by Sottrup-Jensen and collaborators (28). Only three variant residues were found: cysteine vs. glutamic acid at position 563, glutamine vs. glutamic acid at 975, and valine vs. isoleucine at 1000. Residue 975 participates in the formation of the thiol ester bond (28). When the mature protein is sequenced, the bond is hydrolyzed and this residue manifests itself as glutamic acid. However, the cDNA sequence reveals the original resi- due at this position to be a glutamine. Similarly, cDNA se- quence predicted glutamine residues in the thiol ester se- quences of the related murine C3, C4, and sex-limited pro-Table 1. Assignment of the a2M locus to chromosome 12 Human chromosomes Hybrid Human a2M gene 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X laA9.498 ---------------------- Human/rodent somatic cell hybrids tested for the presence of the human a2M gene. References for the hybrids are 1aA9.498, SCC16.5 (gift of D. Cox, Department of Pediatrics, University of California, San Francisco); MOG2, MOG2.1ii (49); HORL9X (50); MCP6 (51); CTP41.2.A2 (52). A human chromosome is scored + if it is present in at least 10%o of cells by karyotyping or the marker enzyme is clearly present by electrophoretic or immunological assay; ± indicates that the marker is present very weakly by enzyme assay. An empty space means not determined by either karyotyping or marker enzyme assay. 
+ + -----+ --------+ -+ MOG2 + + + + + + + + + + + + + + + + + + + + -+ + + DT2.1.2 + -------- + + + + -+ -+ -- + + + SIR7.4ii + + + + -- - -+ + -- ---+ -+ SCC16.5 - HORL9X -
Biochemistry: Kan et al. 
Proc. Natl. Acad. Sci. USA 82 (1985 
) M 6 K N K L L H P S L V L L L L V L L P T D A S V S 26
GCTr ACAATrc CATrcTrGGTrcrCTrCCTAOC TCC r rc TTTTCTrGCAACATGGGGAAGAACAAACTCCT TCATCCAaGTCrGGTT r C TTCTCCTC TTGGTCCTCCTGCCCACAGACGCC TCAGCTC rc 120 
G K P 6 Y N V L V P S L L H T E T T E K 6 C V L L S Y L N E T V T V S A S L E S 66
TGGAAAACCGC AG rATr ATrGGTTrcT TGrCcCrCCCCC TGCTrCCACACTrGAGACCAC TGAGAABGGCTGTGTCCTTCTGAGCTACCTGAATGAGACAB TGAC TGTAAG TGCT TCCT TOGAGTC 240 
V R G N R S L F T D L E A E N D V L H C V A F A V P K S S S N E E V H F L T V a 106
rGTrCAGGGGAAACAGGAGCCTrcTr TCAC TGACCTGGAGGCGGAGAATGACGTACTCCACTGTGTCGCCT TCGCTGTCCCAAAGTCTTCATCCAA TGAGGAGGTAATGTTCCTCAC TGTCCA 360 
V K 6 F T 0 E F K K R T T V N V K N E D S L V F V 0 T D K S I Y K P 6 a T V K F 146
AGTr GAAAGGAC CAAC C CAAGAATrTr TAAGAAGCGGACCACAG T GA TGGT TAAGAACGAGGACAGTC TGGTCT T TGTCCAGACAGACAAATCAATC TACAAACCAGBGCAGACAGTGAAATT r 480 R V V S N D E N F H P L N E L I P L V Y I a D P K G N R I A 0 W 0 S F 0 L E 6 6 
186 TCGTOTTrGrcrCTCCATGGATGAAAAC rrTTCACCCCCTrGAArGAGTrTrGATTCCACTrAOTATACAT TCAGGATCsCAAAGGAAATrCOCATCOCACAATGOCAGAOTTr TCCAOTTrAGAGGGTGG 600 L K 6 F S F P L S S E P F Q 6 S Y K V V V 0 K K S 6 6 R T E H P F T V E E F V L 226 CC T CAAGC AArr TTTCTTT r T CCCCTrcTrCATCAGAGCCCT TC CAGGGC TCCTACAAGGTGGTGGTACAGAAGAAATCAGGTGGAAGGACAGAGCACCCrr TCACCG T GGAGGAATTrrGTT rcr 720 
P K F E V 6 V T V P K I I T I L E E E H N V S V C 6 L Y T Y 6 K P V P 6 H V T V 266 rTC C CAAG rr TTGAAGTr ACAAGT AACAGTr GCCAAAGATr AATCACCAT C rTTGGAAGAAGAGATGAArGrATCASGTBTGTG~CCT ATACACATATCGGAAGCCTGTCCCTGACATGTGAC TGT 840 S I C R K Y S D A S D C H 6 E n s a A F C E K F S 6 a L N S H 6 C F r o 6 V K T 306 GAGCAT T T GCAGAAAGTATAGTGACGCT TCCGACTGCCACGGTGAAGATTCACAGGCTTTCTGTGAGAAATTCAGTGGACAGCTAAACAGCCATGBCTGCTTCTATCAGCAAGTAAAAAC 960 K V F 0 L K R K E Y E N K L H T E A 6 I a E E 6 T V V E L T G R 6 S S E I T R T 346 CAAGGTCTTCCAGC GAAGAGGAAGGAGT ATGAAATGAAACTTCACACTGAGGCCCAGATCCAAGAAGAAGGAACAGTGGTGGAATTACTGGAAGGCAGTCCAGTGAAATCACAAGAAC 1080 I T K L S F V K V D S H F R 0 G I P F F 6 6 V R L V D G K 6 V P I P N K V I F I 386 CAT AACCAACTCTCATTTGTGAAAGT1GGACTCACACrrTCACAGGGAA0TCCCTTCTTTGGGCAGGTGCGCCTAGTAGATGGGAAAGGCGTCCCTATACCAAATAAAGTCATATTCAT 1200 R G N E A N Y Y S N A T T D E H 6 L V 6 F S I N T T N V N 6 T S L T V R V N Y K 426 CAGAGGAAAT GAAGCAAACTAT T ACTCCAATGCT ACCACGGATGAGCATGGCCTTGTACAGTTCTCTATCAACACCACCAACGTTATGGGTACCTC TCTTACTGTTAGGGTCAATTACAA 1320 11 R S P C Y 6 Y a W V S E E H E E A H H T A Y L V F S P S K S F V H L E P N S H 466 GGAT CGTAGTCCCTGTTrACGGCTACCAGTGGGTGTCAGAAGAACACGAAGAGGCACATCACACTGCT TATCT TGTGT TCTCCCCAAGCAAGAGCTTTGTCCACCTTGAGCCCATGTCTCA 1440 E L F C G H T 6 r V 6 A H Y I L N G 6 T L L G L K K L S F Y Y L I N A K G G I V 506 TGAACT ACCC T GTGGCCATACTCAGACAGTCCAGGCACAT T ATAT TCTGAAT GGAGGCACCCTrGCT GGGGCTGAAGAAGCTCTCCTTT TAT TATCTGATAATGGCAAAGGGAGGCATTGT 1560 R T G T H G L L V K a E D N K G H F S I S I P V K S D I A P V A R L L I Y A V L 546
CCGAACTGGGAC TCATGGACTGCT TGTGAAGCAGGAAGACATGAAGGGCCATTTTTCCATCTrCAATCCCTGTGAAGTCAGACATToC TCCTGTCGCTCGGT TGCT CATC TATGCTrGT TTTr 1680 P T 6 D V I G D S A K Y D V E N C L A N K V n L S F S P 5 0 S L P A S H A H L R 586 AGCT AECGGGGACGTBA'TTGGGGAT TCTGCAAAATATGATGTTr GAAAAT TGTCTGGCCAACAAGGTGGAT T TGAGCT TCAGCCCATCACAAAGTCT CCCAGCCTCACACGCCCACCTGCG 1800 
V T A A F Q S V C A L R A V D 0 S V L L N K P D A E L S A S S V Y N L L P E K D 626 AGTCACAGCGGCTCCT CAGTCCGTC TGCGCCCTCCGrGC TGT GGACCAAAGCGTGCTGCTCATGAAGCCTGATGCT GAGCTCTCGGCGTCCTrCGGTT TACAACCTGCTACCAGAAAAGGA 1920 L T 6 F P G P L N D 0 D D E D C I N R H N V Y I N G I T Y T P V S S T N E K D N 666 CCTrCACTGGCT TCCCTOGGGCCTTrTGAATGACCAGGACGAT GAAGAC TGCATCAATCGTCATAATGTCT ATATTAATGGAATCACATATACTCCAGTATCAAGTACAAATGAAAAGGATAT 2040 PrOtsslytic Y S F L E D N G L K A F T N S K I R K P K N C P 0 L 0 0 Y E N H G P E 6 L R V 6 706 GT ACAGCTTCCTAGAGGACATGGGCTrTAAAGGCATTrCACCAACTCAAAGAT TCGTAAACCCAAAATGTGTCCACAGCTrTCAACAGTrATGAAATGCATGGACCTGAAGGTTc ACGTOTAGG 2160 Site F Y E S D V N G R G H A R L V H V E E P H T E T V R K Y F P E T W I U D L V V V 746 r r T TTTATGAGTCAGATGT AATGGGAAGAGGCCATrGCACGCC TGGTGCATGTTGAAGAGCC TCACACGGAGACCGTACGAAAGTACT TCCC TGAGACATGGATCTGGGATT TGGTGGTGGT 2280 N S A G V A E V G V T V P D T I T E W K A 6 A F C L S E D A G L 6 I S S T A S L 786 AAACTCAGCAGGGGTGGCTGAGGTAGGAGTAACAGTcCCTGACACCATCACCGAGTGGAAGOCAGGGGCCTTCTGCCTGTCTGAAGATGCTrGGAC TTGGTATCTCTr TCCACTGCCTCTCT 2400 R A F 6 P F F V E L T N P Y S V I R 6 E A F T L K A T V L N Y L P K C I R V S V 826
CCGAGCCTTCCAGCCCrcTTrTGTGGAGC T TACAATGCC TTr AC TCTGTGAT TCTGGAGAGOCCT TCACAC TCAAGGCCACGGTCCTAAAC TACCTr TCCCAAATGCATCCGGG TCAGTGT 2520 6 L E A S P A F L A V P V E K E 6 A P H C I C A N G R 0 T V S W A V T P K S L 6 866 
GCAGCTGGAAGCCTrcTrCCCGCCTTCCTr TGCTGTCCCAG TGGAGAAGGAACAAGCGCCTCAC TGCATCTGTGCAAACGGGCGGCAAACTGTGTCCTGGGCAGTAACCCCAAAGTCATTAGG 2640 
N V N F T V S A E A L E S 0 E L C G T E V P S V P E H G R K D T V I K P L L V E 906
AAATGTGAAT T TCACT G T GAGCGCAGAGGCACTAGAGTC TCAAGAGC TGrTGTGGGACTGAGGTGCCTr TCAGTTCCT GAACACGGAAGGAAAGACACAGTCATCAAGCC TCTGTTGGTTGA 2760 
P E 6 L E K E T T F N S L L C P S G 6 E V S E E L S L K L P P N V V E E S A R A 946
Thioester Site S V S V 1 6 El I L 6 5 A N 6 N T 6 N L L 6 N P V 6 Cjjjj N N V L F A P N I V V 986 T TC TGT CTC AGT TT T GGG AGACATA T TAGG CT C TGC CAT GCAAAA CACAC AAAAT CT T CTC CAGAT GCCC TAT GGC T GT GAGAG CA-GAATAT GGT C CTCT TTGCT CC TAACATC TA TGT 30G00 
L Dl Y L N E T 6 6 L T P C V K S K A I 6 Y 1 N T 6 V 0 K 6 1 N Y K H V El 6 5 V 5 1026 ACT GG AT T AT CT AAATGAAACACAGCAGCT T AC T CCAGAGGTCAAGT CCAAGGCCAT TGGCTATCTCAACACTGGTTACCAGAGACAGTTGAACTACAAACACTATGAT GGCTCCTACAG 3120 
T F 6 C K V 6 K N 6 6 N T U L T A F V L K T F A 6 A R A Y I F IDEl A H I T 6 A 1066 CACCTT TGGGGAGCGATATGGCAGGAACCAGGGCAACACCTGGCTCACAGCCTTTGTTCTGAAGACTT TTGCCCAAGCTCGAGCCT ACATCT TCATCGATGAAGCACACATTACCCAAGC 3240 L I U L 5 6 K 6 K El N 6 C F R 5 5 6 5 L L N N A I K 6 6 V C El V T L S A Y I T 1106
CC TCATAT GGC TCT CCCAGAGGCAGAAGGACAAT GGC TGT TTCAGGAGCT C TGGGTCAC TGC TCAACAATGCCA TAAAGGGAGGAGTAGAAGATGAAGTGACCCTC TCCGCCTATATCAC 3360 
I A L L E I F L T V T H P V V R N A L F C L E S A W K T A Q E G D H G S H V Y T 1146 CAT CGCCCT TCT GGAGAT TCCT CTCACAGTCACTCACCCTGT TGTCCGCAATGCCCTGT TTTGCCTGGAGTCAGCCTGGAAGACAGCACAAGAAGGGGACCAT GGCAGCCATGTATATAC 3480 K A L L A Y A F A 1 A 6 N 6 El K K K C V L K S L N E C A V K K El N S V H U C K P 1186
C AAAGCAC TGC TGGCC TAT GC TT T TGCCC TGGCAGGT AACC AGGACAAGAGGAAGGAAG TACTCAAGT CACT TAAT GAGGAAGCT GTGAAGAAAGACAACT CT GTCCATT GGGAGCGCCC 3600 
6 K P K A P V 6 H F Y C P 6 A P 5 A C V C N T S V V L L A Y L T A 6 P A F T S E 1226
T CAGAAACCCAAGGCACCAG TGGGGCAT TT T TACGAACCCCAGGCT CCCT C TGCT GAGGTGGAGATGACAT CC TAT GTGC TCCTCGCT TAT CT CACGGCCCAGCCAGCCCCAACCT CBGA 37 20 GGACC TGACC TCT GCAACCAACATCGTGAAGT GGATCACGAAGCAGCAGAAT GCCC AGGGCGGT TT CTCCTCCACCCAGGACACAGTGGT GGCTCTCCATGCT CTGT CCAAATATGGAGC 3840 C GCCACAT T TACCAGGACT GBGAAGGCT GCACAGGT GACT ATC CAGT CT TCAGGGACAT T TTCCABCAAAT TCCAAGTGGACAACAACAAT CGCCTGT TACTGCAGCAGGTC TCAT TGCC 39 60 AGAGC TGCCT GGGGAAT ACAGCATGAAAG TGACAGGAGAAGBAT GTGTC TACCTCCAGACCT CCT TGAAAT ACAATAT TCT CCCAGAAAAGGAAGAGT TCCCCT TTGCT T TAGGAGTGCA 4080 (Legend appears at the bottom of the next page.) 
T L P 0 T C D E P K A H T S F 0 N S L S V S Y T G S R S A S N AM H A I S V K V 13626 S G F I K' L K P A V K M L E R S N H V S R T E V S S N H V L I Y LI 0K V S N P 1426 rTCTGCTCATTCCCGAAGCAAASTGAAAGCTTAAAATCTACCTGTGGCCGACGAAGCAOAGCACCAGTCTGATTACCTGAAAGGOTCAATCG 39602 L S L F F T V L 6 D V P V R D L K P A I V K V Y D Y Y E T D E F A I A E Y
Murine C3 [T]P A S G S Q L -L V LL L L A S S I-L A L G Human A2M M G K N K L L H P S L V L L L L -V L L _ T D A FIG.
2. Amino acid sequence homology between signal peptides of human pro-a2M and murine prepro-C3. The sequences were aligned to give the best homology. The C3 sequence is from ref. 17. 
tein and of human C3 and C4 (17)(18)(19)(20)(21)46). At the 3' end of the cDNA sequence (nucleotide positions 4558-4563) an A-A-T- A-A-A poly(A) addition signal was observed, 89 nucleotides downstream from the translational stop codon and 14 nucde- otides upstream from a stretch of 9 adenines, which appears to be the beginning portion of the poly(A) tail. 
Assignment of the a2M Locus to Chromosome 12. The chro- mosomal locus of the a2M gene was assigned by using so- matic cell genetics. Fig. 3 shows human, mouse, and hybrid DNA digested with Pst I and probed with a nick-translated 1600-base-pair cDNA fragment representing the 3' proximal portion of human a2M mRNA. The human DNA in track 1 shows restriction fragments of 16, 5.8, 5.5, 4.3, and 2.5 kb, whereas the mouse DNA in track 10 shows major restriction fragments of 9.5, 6, and 5.5 kb. Three low-abundance mouse fragments are seen in some of the hybrids at 19, 14, and 3.5 kb. Hybrids in tracks 2, 4, and 9 are positive for the diagnostic human fragments. Track 4 contains DNA from the hybrid cell line laAq.498, which contains human chromosomes 6, 12, 21, and X. Hybrid cell line SCC16.5 (track 5) is negative and contains human chromosomes 21 and X. Hybrids MCP6 (track 8) and CTP41.2.A2 (track 6) are also negative and con- tain chromosome 6, among others. These combined results indicate that the human a2M gene is located on chromosome 12. Table 1 presents all of the hybrids tested and their chromosome compositions. The only chromosome showing com- (48). DNA was di- gested with Pst I and 25 ,ug of DNA was loaded per track. The + and -refer to the presence and absence of diagnostic human fragments. 
plete concordance with the a2M gene is 12. The other chro- mosomes show at least two discordancies. Identical results were obtained when the same hybrids were digested with the restriction nuclease Taq I. We therefore conclude that the a2M gene is located on chromosome 12. 

DISCUSSION

a2M cDNA clones were isolated in this study by using two synthetic oligonucleotide probes representing neighboring regions in the protein, located in its carboxyl-terminal por- tion. Successive screening with both probes eliminated the problem of false positives. One cDNA clone was found to contain an insert with the entire coding sequence for the a2M protein, including its signal peptide. To date, no amino acid sequence data are available to confirm the signal peptide se- quence predicted from cDNA; however, the sequence shows substantial similarity with the corresponding human and mu- rine C3 signal peptides. The amino acid sequence derived from cDNA confirms the protein sequence reported by Sot- trup-Jensen and co-workers (28) in all but three residues. 
The glutamine/glutamic acid difference in the thiol ester site (residue 975) has been explained in Results. The finding of a glutamine further supports our proposal that the formation of the thiol ester is initiated with a glutamine rather than a glutamic acid (46,53). The difference between a cysteine and a glutamic acid residue at position 563 raises a question about the reported disulfide-bridge structure of the molecule (28). 
Further sequence analysis of this region of the protein would be desirable. The third difference, valine vs. isoleucine at position 1000, might be due to a DNA polymorphism, be- cause the corresponding coding triplets can be converted from one to the other by a single base mutation resulting in a conservative replacement between two functionally similar amino acids. 
The a2M gene belongs in a gene family together with the complement C3, C4, and C5 genes. In mice, the C3 and C4 genes reside on the same chromosome, chromosome 17 (54); in humans they reside on different chromosomes: C3 on chromosome 19 (23) and C4 on chromosome 6 (24). Thus, no selective pressure is apparent to maintain these loci on the same chromosome after duplication and divergence. The as- signment of the human a2M locus to yet a different chromosome suggests that no selective pressure keeps the C3, C4, and a2M loci together on the same chromosome. 
Frotn the data presented here we cannot definitively eval- uate the number of a2M loci on chromosome 12. The possi- bility exists that more than one a2M locus or cross-hybridizing loci might be present on chromosome 12, in analogy to the duplicated C4 loci on chromosome 6. Rats and rabbits have two a-macroglobulins in their serum: a1-macroglobulin (a1M) and a2M (4). Both are similar in size, are proteinase inhibitors, contain thiol ester bonds, share antigenic determinants, and show similar amino acid composition and glyco- sylation patterns (4). Thus, most likely they are products of duplicated genes. For humans, an equivalent of the ajM pro- tein has not been found. We had anticipated that an a1Mrelated gene might be detected by cross-hybridization in the FIG. 1 (on preceding page). cDNA and deduced amino acid sequences of pa2Ml, showing the 5' nontranslated sequence, the signal peptide, the entire a2M protein sequence, the poly(A) addition signal, and a portion of the poly(A) tail. The standard one-letter code for amino acids ( 45) is used. The proteolytic cleavage site and the thiol ester site are underlined. Also shown are regions of nucleotide sequences from which the 14- mer and 17-mer oligonucleotide probes were derived (broken underlines). 
human genome, but until now we have not found its trace. 
The cDNA sequences reported here can be used for isola- tion of the corresponding rat and human a2M genomic DNA clones and for further studies of the control of expression of these genes during acute inflammations (55). 



  1378 cy. These amino acid sequences are Asn-Met-Ala-Ile-1385 Val-Asp and Asp-Val-Lys-Met-Val. The corresponding oligonucleotide mixtures were AA ST'-T-C-N-A-C-G-A-T-N-G-C-C-A-T-G _T-T-3' 




  + MCP6 -. . . . . .+. . . . . . . . . . . . . . . .+ MOG2.1ii -+ -+ + + + -+ + -+ -+ + + + + + + + + -+ CTP41.2.A2 --+ --++ ----+ ----+ 


Fig. 1 .

 1 Fig. 1. (Legend appears at the bottom of the next page.) 


FIG. 3 .

 3 FIG.3. Detection of human a2M gene sequences in human/ mouse somatic cell hybrids. Track 1, human (HU) line Molt-4 (47); track 2, hybrid (hy) MOG2; track 3, hybrid MOG2.1.ii; track 4, hy- brid laA9.498; track 5, hybrid SCC16.5; track 6, hybrid CTP41.2.A2; track 7, hybrid Horl9X; track 8, hybrid MCP6; track 9, hybrid DT2.1.2; track 10, mouse (M) line IR (48). DNA was di- gested with Pst I and 25 ,ug of DNA was loaded per track. The + and -refer to the presence and absence of diagnostic human fragments. 




  line Molt-4 (47); track 2, hybrid (hy) MOG2; track 3, hybrid MOG2.1.ii; track 4, hy- 



brid laA9.498; track 5, hybrid SCC16.5; track 6, hybrid CTP41.2.A2; track 7, hybrid Horl9X; track 8, hybrid MCP6; track 9, hybrid DT2.1.2; track 10, mouse (M) line IR 
Proc. NatL. Acad. Sci. USA 82(1985) 

We thank Dr. Lars Sottrup-Jensen for making the a2M sequence available prior to publication, Dr. Maarten de Bruijn for making C3 data available and for helpful discussions, Dr. Hans Muller-Eber- hard for support, Dr. Michael C. Carroll and Prof. Rodney R. Porter for their cooperation, and Keith Dunn for preparation of the manu- script. This work was supported by Research Grant AI19651 from the National Institute of Allergy and Infectious Diseases. This is publication no. 3597IMM from the Research Institute of Scripps Clinic.
Chen-Chen Kan, Ellen Solomont, K Tertia Beltt, Aileen C Chain, Lynne R Hiornst, Georg Fey
Nucleotide sequence of cDNA encoding human a2-macroglobulin and assignment of the chromosomal locus (human serum proteins/proteinase inhibitors/human chromosome mapping/thiol ester bond)