Abstract
It has been shown that the relative reaction preference of the C4 thiol ester toward oxygen and nitrogen nucleophiles upon activation by proteinase depends on whether residue 1106 is aspartate or histidine (Dodds, A. W., Ren, X.-D., Willis, A. C., and Law, S. K. A. (1996) Nature 379, 177-179). To determine if the equivalent residue in the related thiol ester-containing protein human alpha2-macroglobulin (alpha2M), asparagine 1065, plays a similar role, we have expressed and characterized four alpha2M variants in which this asparagine has been replaced by aspartate, alanine, histidine, or lysine. The change from asparagine resulted in an altered ability to form the thiol ester. This ranged from failure to form the thiol ester (Asn --> Asp) to a maximum extent of formation of about 50% (Asn --> Ala). For the three variants that were able to form the thiol ester, the rates of thiol ester cleavage by a given amine were found to be different from one another and slower in nearly all cases than plasma alpha2M, but with the same relative reactivity of methylamine > ethylamine > ammonia. The rate of conformational change that follows cleavage of thiol esters in a functional half-molecule was also found to differ between the variants and to be slower than plasma alpha2M. TNS emission spectra indicated that the conformations of the transformed variants differed measurably from transformed plasma alpha2M. These findings suggest that residue 1065 plays a critical role in human alpha2M, for formation of the thiol ester, for its subsequent reaction with nucleophiles, and for the conformational change induced by this reaction. By analogy with C4, where this residue influences the nucleophile preference through direct interaction with the thiol ester, residue 1065 in alpha2M is expected to be located in or very close to the thiol ester region in alpha2M.
Highlights
The abundant human plasma protein ␣2-macroglobulin (␣2M)1 shares a number of properties with the two complement proteins C3 and C4
C3 shows a preference for oxygen nucleophiles [2], whereas C4 shows a preference for nitrogen nucleophiles, but with an increase in reactivity toward oxygen for the B isotype [3, 4]
We set out to determine whether residue 1065 of human ␣2M plays a role in the reactions of the mechanistically important thiol ester by creating four ␣2M variants with single site mutations at this position and characterizing the properties of the expressed proteins
Summary
The abundant human plasma protein ␣2-macroglobulin (␣2M) shares a number of properties with the two complement proteins C3 and C4. More definitive information on the molecular basis for the nucleophile preference of the thiol ester in C4 has been provided by an examination of the effect of single site mutations at position 1106 of C4 This is a position 115 residues C-terminal from the cysteine that is involved in forming the thiol ester, it is one of only four residues, occurring in a hexapeptide, that differ between the C4A and C4B isotypes of C4. Comparison of all available sequences of ␣-macroglobulins (13 total) showed that asparagine occurs in 11 and histidine in 2 (Table I) Because of this high conservation of asparagine and the presence of histidine as the only alternative, as in C4B, we sought to determine whether this residue influenced the properties of the thiol ester in human ␣2M.
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