The primary sequence and the subunit structure of mouse α‐2‐macroglobulin, deduced from protein sequencing of the isolated subunits and from molecular cloning of the cDNA

Abstract

Mouse plasma α‐2‐macroglobulin (mα2M) was isolated and the N‐terminal amino‐acid sequences determined after separation of the 165‐kDa and 35‐kDa subunits. These sequences were compared to the protein sequence predicted by the cDNA, which was cloned from a mouse liver library and sequenced. From these data it is evident that both subunits are encoded by one mRNA of approximately 5kb expressed predominantly in liver. The smaller subunit, with the N‐terminal sequence DLSSSDLT, comprises the C‐terminal 257 residues of mα2M and is derived from a single‐chain precursor probably by proteolytic processing at an arginine residue in the sequence PTRDLSS. Analysis of the predicted protein further showed all the salient features of a proteinase inhibitor of the macroglobulin family: a bait region that deviates from all known sequences in this family, a very conserved internal thiolester site and conserved cysteine residues and putative N‐glycosylation sites. The synthesis of mα2M in adult liver was demonstrated by Northern blotting and in fetal liver by in‐situ hybridization. Transient transfection of COS cells with the cDNA under control of a viral promoter demonstrated the secretion and partial processing of mα2M in the culture medium. In plasma the level of mα2M was found to be stable as expected for the murine counterpart of human plasma α‐2‐macroglobulin. The possibilities of using the mouse as a genetic model to study this proteinase inhibitor in vivo are discussed. Copyright © 1992, Wiley Blackwell. All rights reserved