Meprin A and meprin B are disulfide-linked, oligomeric metalloendopeptidases in
renal brush border membranes. Meprin A contains 90-kDa subunits (α subunits) and is
expressed in random-bred and some inbred strains of mice. Meprin B contains subunits of
110 kDa (β subunits) in situ, and the enzyme from C3H1He mice, a strain that does not
express α subunits, has been characterized. Evidence from this and previous studies
indicate that β subunits are expressed in all mouse strains. Meprins were characterized
with regard to their glycosylation by lectin blotting. Both meprin A and meprin B bound
the lectins concanavalin A and the erythroagglutinin from Phaseolus vulgaris indicating
that both enzymes contain high mannose and bisected biantennary complex type
oligosaccharides. However, meprin A, but not meprin B, bound the agglutinins from
Ricinus communis, Datura stramonium, and the leukoagglutinin from Phaseolus vulgaris,
indicating that complex-type N-glycosylation differs in these proteinases. Lectin blots of
membrane proteins from C57BL/6 mice indicated that there were differences between
adult male and female mice in the glycosylation (specifically in the complex type
oligosaccharides) of the α subunit of meprin A. A marked degree of carbohydrate
heterogeneity was observed in meprin A from males as compared to the enzyme isolated
from female mice. Additionally, the data indicated that at least three of the ten potential
glycosylation sites in the meprin α subunit are glycosylated. Overall, these studies expand
our understanding of how estrogens affect glycosylation of meprin A. The oligomeric
organization of the meprins was examined in brush border membrane fractions from a
random-bred strain (lCR) and two inbred strains of mice (C57BL/6 and C3H1He). The
random-bred strain contained three oligomeric complexes of approximately 390, 440, and
490 kDa as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-P AGE) in the absence of reducing agents. The subunits in all three oligomers were
linked by disulfide bridges. Western blotting using anti-a monoclonal antibodies revealed
that α subunits (90 kDa) were present in the 390- and 440-kDa complexes. Western
blotting with polyclonal antibodies specific for the β subunit (110 kDa) revealed the
presence of these subunits in the 440- and 490-kDa complexes. Electroelution of the
individual oligomers followed by SDS-PAGE under reducing conditions confirmed that
the 390- and 490-kDa molecules are homotetramers of α and β subunits, respectively, and
that the 440-kDa complex is a heterotetramer composed of disulfide-linked α and β
subunits. C57BL/6 mice expressed both α and β subunits and contained tetramers
composed of u4 and U 2β2. C3H1He mice expressed only the 110-kDa β subunits and the
β4 oligomer. This type of multimeric organization of covalently-linked subunits is unique
for the known endopeptidases. Initial cloning of the mouse meprin β subunit revealed that
the enzyme belongs to the recently described astacin family of metalloendopeptidases.
The β subunit polypeptide had a molecular mass of 88 kDa as determined after SDSPAGE
of brush border membrane proteins treated with glycosidases. Nucleotide
sequencing, internal peptide sequences from the β subunit, and NH2-terminal sequence
analyses (3 9 residues) indicated that at the amino acid sequence level, mouse β is
approximately 55 % identical to mouse α, and 85 % identical to the rat β subunit. These
and other studies indicate that α and β are closely related products of divergent evolution.
Northern blot analyses of different tissues from C57BL/6 and C3H1He mice indicate that β
subunit mRNA can be detected in kidney and intestine, in contrast to the u subunit which
is only present in kidney tissue. Initial studies in mouse intestinal brush border membranes
indicated that the characteristic latency of kidney β subunits may be absent in the intestinal
enzyme. This observation may reflect activation of the mouse β subunit by trypsin in the
intestinallumen. The activation of β in the kidney by trypsin-like proteinases is
reminiscent of the activation of protein zymogens and may serve as a means of regulation
of the proteolytic activity of the proteinase at the cell surface.
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