MDC-L, a Novel Metalloprotease Disintegrin Cysteine-rich Protein Family Member Expressed by Human Lymphocytes

Abstract

The metalloprotease disintegrin cysteine-rich (MDC) proteins are a recently identified family of transmembrane proteins that function in proteolytic processing of cell surface molecules and in cell adhesion. Since lymphocytes must interact with a constantly changing environment, we hypothesized that lymphocytes would express unique MDC proteins. To identify MDC proteins expressed in human lymph node, a polymerase chain reaction-based strategy combined with degenerate oligonucleotide primers was employed. We report here MDC,1 or ADAMs, are a family of transmembrane glycoproteins with a unique domain structure (1, 2). The prototypical MDC protein comprises pro, metalloprotease, disintegrin, cysteine-rich, EGF, transmembrane, and cytoplasmic domains. The metalloprotease domain is homologous to the reprolysins, members of the zinc binding metzincin superfamily that also includes the matrix metalloproteases (MMPs), the astacins, and serralysins (3). The prodomain regulates the activity of the metalloprotease by blocking access to the zinc ion; removal of the prodomain or inactivation of a conserved cysteine thiol group results in a gain of proteolytic activity. The disintegrin domains of MDC proteins are homologous to small nonenzymatic peptides isolated from the venom of snakes. Snake venom disintegrin peptides interfere with platelet aggregation by inhibiting binding of fibrinogen to the integrin ␣ IIb ␤ 3 (4). The functions previously attributed to the individual domains suggests roles for MDC proteins in cell adhesion and the proteolysis of extracellular proteins.The first mammalian MDC proteins identified were the fertilins (5). These proteins function in the attachment and fusion of the sperm and egg during fertilization. A similar cell fusion function for the MDC protein meltrin-␣ (MDC-12) was shown for the formation of multinucleated myotubes (6). These MDC proteins have, in addition to the domains described above, a fusion peptide-like sequence not found in all members of the MDC protein family. However, the interaction of the fertilin disintegrin domain on the sperm surface with the integrin ␣ 6 ␤ 1 on the surface of the egg is also required for membrane adhesion and fusion (7-9). Additional evidence for the functionality of the disintegrin domain comes from studies using recombinantly expressed metargidin (MDC-15) disintegrin domain, which was shown to specifically interact with the integrin

Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28

“…For epididymal RNA, the probe 2065-2410 recognized an abundant 2.9 kb transcript and a lower level of a 4.3 kb transcript, but not the 2.3 kb transcript recognized by the probe 1246-1613 ( Figure 2C). These data suggest that mouse ADAM28 RNA exists in at least two splice forms in the epididymis, and thus resembles the two splice forms of human ADAM28 in lymphocytes, MDC-Lm and MDC-Ls [35]. The 2.9 kb and 4.3 kb transcripts are predicted to encode membrane-anchored forms of ADAM28, whereas the 2.3 kb transcript, which is not detected in the lung, presumably encodes a secreted form lacking the membrane-spanning and cytoplasmic domains.…”

“…To analyse ADAM28 expression, RNA was isolated from mouse lung and epididymis [34]. To look for alternative transcripts [35], duplicate RNA samples (25 µg for each tissue) were examined by Northern blotting using a probe corresponding to bases 1246-1613 (encoding part of the metalloprotease-and disintegrin-like domains) or bases 2065-2410 (the region encoding the transmembrane and cytoplasmic domains). A [$#P]dCTP-labelled human β-actin cDNA probe (Clontech) was used as a control for RNA loading.…”

“…macaque eMDC II ( Figure 1) and a polypeptide encoded by a rat EST (expressed sequence tag) (AI030215) [35,38]. This protein is designated ADAM28.…”

The Lymphocyte Metalloprotease MDC-L (ADAM 28) Is a Ligand for the Integrin α4β1

“…The HepG2, a hepatocellular carcinoma cell line, was maintained in Eagle's modified essential medium containing 10% fetal calf serum, 1% L-glutamine, and 1% penicillin/streptomycin. The ␣ 4 -transfected K562 cell line was a generous gift from Dr. R. Isberg, Tufts University School of Medicine, Boston, MA (47 (31). The purified MDC-L fusion proteins were mixed and used as immunogens.…”

“…MDC-L (ADAM 28) is a member of the ADAM protein family possessing a prototypical domain structure (31,32). MDC-L is predominantly expressed on the surface of human lymphocytes (31); however, others have also reported expression within mouse epididymis (32). The metalloprotease domain of MDC-L possesses catalytic activity, as demonstrated by the ability of MDC-L to cleave the substrate myelin basic protein (33).…”