Primary Structure of Human a2-Macroglobulin COMPLETE DISULFIDE BRIDGE ASSIGNMENT AND LOCALIZATION OF TWO INTERCHAIN BRIDGES IN THE DIMERIC PROTEINASE BINDING UNIT*

The disulfide bridge pattern of human az-macroglob-ulin (azM) given earlier (Sottrup-Jensen, L., Stepanik, T. M., Kristensen, T., Wierzbicki, D. M., Jones, C. M., Lmblad, P. B., Magnusson, S., and Petersen, T. E. (1984) J. Biol. Chem. 259, 8318-8327) has been re-vised by showing that CyszSs and Cys408 in one subunit are bridged to Cys408 and CysZB6, respectively, in the adjacent subunit of the proteinase binding dimer. Thus, the azM-dimer contains two interchain disulfide bridges, and the individual subunits are arranged in an antiparallel fashion. These results are the outcome of partial reduction experiments, where reduction of methylamine-treated azM with 1-8 mM mercaptoeth- anesulfonate at pH 8.0 resulted in the appearance of 2.6 mol of SH-groups per mol of free subunit. Apart from reduction of the two interchain bridges, the intrachain bridges Cysz28-Cys276, C y ~ ~ ’ ~ - C y s ~ ~ ~ , CysszS, and Cys8z4-Cys8so are reduced to a minor extent under these conditions. The disulfide bridge pattern of azM has been completed by showing that the azM subunit contains 11 intrachain bridges, including a bridge connecting Cys447 with CYS’~~.

The primary structure of the human plasma glycoprotein a2M1 has been determined recently by methods of protein chemistry (1)(2)(3)(4)(5). The identical 180-kDa subunits of a2M contain 1451 residues and are pairwise disulfide-bridged to 360-kDa dimers (6,7). These are further noncovalently associated to the tetrameric a2M of 720 kDa. a2M forms stable complexes with a variety of proteinases from all four classes, EC 3.4.21-24, and complex formation is initiated by specific limited proteolysis in the so-called "bait" region, residues 681-686 (5). azM .proteinase complexes are rapidly cleared from the circulation by receptor-mediated endocytosis, primarily by hepatocytes and Kupffer cells of the liver. The disulfidebridged 360-kDa dimers of azM constitute its functional proteinase binding units (8)(9)(10)(11). The localization of 11 disulfide bridges in a2M (5) was unambiguously determined by the * This work was supported by grants from the Novo Foundation and the Danish Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence and requests for reprints should be addressed.
"performic acid diagonal technique" using small peptides generated from sets of disulfide-bridged CNBr fragments (1)(2)(3)(4). Since Cysg4' presumably is thiol-esterified to Glxg5', the single remaining half-cystine residue at position 447 was thought to form an interchain disulfide bridge to Cys447 of another subunit (5). However, this could not be proven, and the recent finding that residue 540 is a half-cystine not only in a2M (12, 13), but also in the strongly related human pregnancy zone protein (14,15), evidently questioned this proposal. This further indicated that Cys447 and might be disulfidebridged, and, moreover, that the number of interchain disulfide bridges in the covalently bound dimeric unit of aZM must be two or a multiple thereof (12).
Here we complete the primary structure determination of human a2M by showing that Cys255 and Cys40s, previously thought to form an intrachain disulfide bridge in the 180-kDa subunit of a2M in fact are engaged in the formation of two interchain disulfide bridges within the 360-kDa azM-dimer, and that Cys447 forms an intrachain bridge with

RESULTS
Partial Reduction of a2M. MA with MES-No information on the localization of interchain bridges in azM can be obtained from analysis of small disulfide-bridged peptides, since these could represent either intra-or interchain bridges. As seen from the alignment of the CNBr fragments shown in Fig. 1, none of the disulfide bridges within CB2, CB6, CB18, and CB21 can be interchain bridges, since this would be revealed by a characteristic change in size when examined by nonreducing and reducing SDS-PAGE. Under nonreducing conditions, the size of CB2 was estimated at 18 kDa, while those of CB6 + CB8, CB18 + CB23 + CB26, and CB21 were 20,40, and 28 kDa, respectively (1). Rather than investigating conditions where sufficiently large fragments could be generated from the a2M-dimer so that the assignment of interchain bridges could be based on SDS-PAGE analysis, partial reduction of a2M was investigated instead. Pilot studies using 1 mM dithioerythritol at pH 8.0 indicated reduction of about six to eight disulfide bridges for nearly complete disappearance of the 360-kDa disulfide-bridged dimer of native azM (not shown), compatible with earlier results (9,21). Since the interchain bridges presumably are among the most solvent-accessible and hence most easily reduced bridges, their selective reduction was investigated using mercaptoethanesulfonate (MES). This reagent is strongly solvated and has a higher redox potential than that of dithioerythritol (22), and might yield a more selective reduction than dithioerythritol. Analytical time course experiments using 6 mM MES at pH 8.0 indicated that for native a2M there was a progressive reduction of interchain bridges, whereas for a2M. MA, no further reduction appeared to take place after about 10 min as judged from SDS-PAGE under nonreducing conditions (not shown). Accordingly, the reduction of a z M -MA with varying amounts of MES was investigated. Fig. 2A shows the appearance of the 180-kDa subunit as a result of incubating a2M -MA with increasing amounts of MES (0.4-32 mM) for 30 min a t room temperature. In Fig. 2B, the generation of partially reduced a2M.MA subunits is related to the total amount of subunit present. In parallel, the amount of SH-groups present in partially reduced a2M. MA was determined by DTNB titration after removal of excess reductant by gel chromatography and related to the amount of subunit present. The results demonstrated a close correlation between the SH-groups appearing and the extent of generation of partially reduced subunit. In the range of 1-8 mM MES, the stoichiometry between SH-groups appearing and partially reduced subunits appearing was close to 2.6. At higher levels of MES, excessive reduction took place, but even at the highest concentration of MES used (32 mM), only about 83% of the subunits had their interchain bridges reduced. Under the same conditions, the degree of reduction of disulfide bridges in native azM was higher (Fig. 2B), but even at a point where about 5.5 SH-groups had appeared per subunit (at 14 mM MES), 50% of the a2M dimers still had their interchain bridges intact as judged from nonreducing SDS-PAGE (not shown). Hence, a t low concentrations of MES, the generation of the partially reduced subunits of azM ' MA is dependent on the reduction of only two interchain bridges in the 360-kDa dimer, accompanied by a minor extent of reduction of other (intrachain) disulfide bridges.
Localization of the Interchain Disulfide Bridges in the a2M Dimer-On a preparative scale, a2M. MA was reduced with 4 mM MES for 10 min and the SH-groups appearing labeled by reaction with tritium-labeled iodoacetic acid. After exhaustive reduction with dithioerythritol and carboxymethylation using unlabeled iodoacetic acid in the presence of guanidinium chloride, chymotryptic peptides were generated and separated on a DEAE-Sephacel column (Fig. 3, Miniprint). Four pools containing the major part of the radiolabel were further separated by ion-exchange and reverse-phase HPLC (Figs.  (2), this result implies that Cys255 and Cys408 in one subunit are bridged to Cys408 and CYS'~~, respectively, in the adjacent subunit of the aZM-dimer (Fig. 1).
Identification of Internal Disulfide Bridges in a2M. MA Reduced to a Minor Extent by MES-Investigating a number of minor peaks of radioactivity obtained from the initial reversephase HPLC separations (Miniprint) sequence analysis revealed the presence of chymotryptic peptides containing the following Cys residues: CyP6, CYS~~', CysSz4, and CysSZ6 (Fig. 7, Miniprint). The incorporation of tritium label in these (impure) peptides was estimated a t 0.2-2.2 x lo3 cpm/nmol as opposed to 10-12 X IO3 cpm/nmol for the major peptides CT1-CT4. This result indicates that the intrachain bridges Cys228-Cys276, C y~~~~-C y s~~, C y~~'~-C y s~~ and, in particular, are relatively exposed to solvent. Completion of the Disulfide Bridge Assignment of a2M-Since C Y S '~~ a n d C~S~'~ are engaged in interchain bridge formation, C Y S "~~ and CYS~~O would be expected to form an intrachain bridge. These residues are located in CB9 and CB11, respectively, and a pool containing the medium sized CNBr fragments from a2M was isolated (Figs. 8 and 9, Miniprint). After reverse-phase HPLC on a Vydac C4 column (Fig. lOA, Miniprint), the expected fragment set was recovered pure, as judged from amino acid analysis and sequence determination (Tables 2 and 3, Miniprint). This strongly indicated that CB9 was disulfide-bridged to CB11, which is further bridged to CR17 (2,5). Following reduction, "C carboxymethylation, and rechromatography on the same column (Fig. 10B, Miniprint ), CB9, CR11, and CB17 were isolated. While amino acid analysis ( Table 2, Miniprint) clearly showed that the fragments indeed were CB9, CB11, and CB17, CB17 was recovered in a very low yield as also seen before (1,2). For unknown reasons, the NH2 termini of these fragments had become blocked during isolation, and no verification could be made by sequence analysis. The bridge connecting Cys447 and Cys"'"' was finally proven by analyzing a chymotryptic digest of CR9 + C B l l + CB17. The peptides were separated by reverse-phase HPLC, and the relevant bridge peptide(s) were identified following reduction and "H carboxymethylation (Fig. 11,A and B, Miniprint). The results of amino acid analysis and sequence determination of the peptides represent.ing the bridges Cy~~~'-Cys"" (CT5 and CT6) and Cys'jR (CT7 + CT8) are shown in Table 4 and Fig. 12 (Miniprint), respectively.

DISCUSSION
In continuation of earlier investigations on the primary structure of human cu2M (l-s), we have now completed the determination of its disulfide bridge pattern. The subunit. of a2M contains 11 intrachain bridges, including a bridge connecting Cysj4' and the recently recognized Cys"" (12. 13). The subunits of the proteinase binding rupM-dimer are held together by two interchain bridges. These bridges connect Cys"" and C~S " "~ in one subunit with C~S ' " '~ and Cy?", respectively, in the adjacent subunit. Cys".' and Cys4"" were known from previous studies to be disulfide-bridged (2,5), and the assignment of these half-cystine residues as being involved in interchain formation was the outcome of partial reduction experiments. Although not investigated in detail, a minor extent of reduction in cu2M.MA of the following int.rachain bridges could be demonstrated: CysF2'-Cys2'', Cy~"~-Cys'.'', cys;"" Cys*16, and Cy~~"'-Cys~"~. Of these bridges, Cy~'!'~--Cys~''' was most easily reduced.
The use of cu,M. MA in these experimenh was important for localizing the interchain bridges since these bridges were more easily reduced in cu2M.MA than in native cu2M (21). In addition, reduction of interchain bridges in CY." .MA appeared to be complete in about 10 min at 6 mM MES, while a time-dependent reduction of interchain bridges in native ( Y~M was seen, accompanied by additional reduction of other bridges (Fig. 2B). Thus, the conformational change accompanying thiol ester cleavage by methylamine results in an increased accessibility of the interchain bridges. While HPLC gel chromatography and nondenaturing PAGE revealed the presence of free subunits, dimers, and intact tetramers upon reduction of native aZM with MES, as also found earlier with N-acetylcysteine (6), partially reduced N?M. MA contained only tetramers (not shown). Thus, cu2M.MA forms a very stable tetramer even when the interchain bridges have been reduced (9). Although extensive reduction of native cu2M results in subunits which readily form insoluble aggregates and thus presumably are denatured ( G ) , reduct,ion of only four to eight disulfide bridges per dimer does not change the tertiary structure of the subunit of native cu?M to any great extent (9,21). In one case, active tetramers were recovered upon reoxidation (9), while noncovalently associated active dimers were obtained in another case (21). In both instances, the internal P-cysteinyl-y-glutamyl thiol esters were largely intact.
The stretches around Cys'" and C~S.'"~ are strikingly hy- subunit has yet been obtained. The individual chains of t.he dimeric unit of cu2M are arranged in an antiparallel fashion (Fig. 1, inset). This orient,ation would be compatible with that proposed in a recent hypot.hetica1 model of cu2M primarily based on electron microscopy and x-ray scat.tering studies (24). In this model, the proteinase binding disulfide-bridged n2M-dimer is regarded as a lobed basket-or mesh-like structure, capable of accommodating one proteinase molecule within its lumen. Whether the partially reduced noncovalently associated dimers re-ported in Ref. 21 represent the disulfide-bridged dimers of a2M, or whether they represent dimers assembled from subunits not covalently linked in the a2M tetramer is presently unclear.
In view of the common evolutionary origin of a2M and the complement proteins C3 and C4 (25), it is likely that the a2M subunit contains a number of functional domains shared with C3 and C4. However, the disulfide bridge pattern of those proteins, which circulate as proteolytically processed "monomers," is markedly different from that of a2M. The existence of two interchain bridges in human a2M, and probably also human pregnancy zone protein and most other azMs, is a unique feature, presumably reflecting the tight association of two subunits necessary for formation of the functional dimeric proteinase binding unit. In this respect, murinoglobulin (26) and rat a1 inhibitor 111 (27) form a n exception since they circulate as monomers or perhaps as noncovalently associated dimers. Following cleavage of only one bait region in a2M, the subunits express co-operativity with regard to activation of the internal thiol esters, so that two SH-groups appear for binding of one proteinase molecule under conditions of low proteolytic activity (11). The ensuing conformational change, which results in irreversible binding and effective steric shielding of the activating proteinase, also appears to be a property of the covalently associated dimer.