Molecular characterization of the murinoglobulins

Proteinase inhibitors of the alpha 2-macroglobulin (alpha 2M) type, although well characterized in vitro, still evade a precise description of their actual role in vivo. The main reason for this is the absence of any clinical evidence for the malfunctioning of alpha 2M in humans. Moreover, despite their ubiquitous presence in animals of very different taxa, animal models are notoriously absent in this field. With the advent of transgenic animals an important tool became available in this respect. As a first step in this direction we are analyzing at the molecular level all the members of this proteinase inhibitor family in the mouse. To retrieve related sequences we screened a mouse liver cDNA library with human alpha 2M cDNA. The sequences from two isolated clones partially coded for a protein with a high degree of sequence identity with human alpha 2M, rat alpha 2M, and rat alpha 1I3. Protein sequence data from the large and small subunits of mouse alpha 2M and of the protein isolated from mouse plasma allowed us to designate the clones as coding for murinoglobulin (MUG), an alpha 2M-related single-chain proteinase inhibitor. Rescreening resulted in the isolation of 24 clones, of which 21 were related or identical to the original MUG clones. Restriction analysis led to three groups of clones of which representative members were sequenced. Two highly homologous cDNA sequences were derived, coding for proteins that displayed the typical features of alpha 2M-type proteinase inhibitors: the overall size, the positions of a putative bait region and of the internal thiol ester, and the positional conservation of cysteine residues and putative asparagine-glycosylation sites. A third related member for which only one incomplete cDNA clone was obtained and sequenced, proved to be aberrant: the bait region contained what appeared to be an intron which escaped proper splicing. A second apparent intron was present at the 5' end of the cDNA while a frameshift mutation near the 3' end (insertion of a G) caused premature termination of the reading frame when compared to the other MUG sequences. These features were confirmed from an isolated genomic clone and extended at the genomic level: the corresponding gene, a transcriptionally weakly active pseudogene, contained the small intron but as part of a larger intron. The presence of suitable intron/exon splice sites show that a relatively small part of the intron is being introduced as an exon in the mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)