Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that play a role in fertilization, myoblast fusion, neuronal development, and cleavage of the membrane-anchored cytokine tumor necrosis factor-␣. Here, we report the cloning and cDNA sequencing of the mouse metalloprotease disintegrin MDC15 and an analysis of its processing in the secretory pathway. A notable difference between mMDC15 and its putative human orthologue (hMDC15, metargidin) is the presence of the peptide sequence TDDC instead of the RGDC found in the disintegrin domain of hMDC15. In a Western blot analysis the majority of mMDC15 was found to lack the pro-domain in all mouse tissues examined. Pulse-chase experiments in transiently transfected COS-7 cells suggest that mMDC15 is processed by a pro-protein convertase in a late Golgi compartment, since (i) addition of brefeldin A or monensin blocks prodomain removal, (ii) all detectable processed mMDC15 is endoglycosidase H -resistant, and (iii) a recombinant soluble form of the trans-Golgi network pro-protein convertase furin can mimic mMDC15 processing in vitro. Cell-surface trypsinization revealed that more than half of mature mMDC15 is intracellular. Immunolocalization provided evidence for a strong perinuclear accumulation in a region resembling the trans-Golgi network and/or endosomal compartments. This study provides the first characterization of the intracellular processing of a metalloprotease disintegrin, and highlights the potential role of pro-protein convertases in removal of the inhibitory pro-domain. These results further suggest possible intracellular functions for mMDC15, such as in protein maturation, in addition to a potential role in cell-surface proteolysis or cell adhesion.Proteolytic processing in the secretory pathway is often a crucial step in the maturation and activation of secreted and membrane-anchored proteins. Pro-protein convertases such as furin play critical roles in protein maturation and have been implicated in the cleavage and activation of several different types of proteins, including pro-hormones (1-3), viral fusion proteins (4 -7), integrin ␣ chains (8), transforming growth factor-␤ (9), the human insulin proreceptor (10), and specific membrane-anchored matrix-type metalloproteases (11,12). In the case of the matrix metalloprotease stromelysin 3, intracellular proteolytic processing of the zymogen by furin results in activation of the protease (11). Several members of a different family of metalloproteases, the metalloprotease disintegrins, also contain cleavage sites for pro-protein convertases in their extracellular domain. This suggests that proteolytic processing in the secretory pathway plays a role in the maturation and activation of these proteins (13-15).Metalloprotease disintegrins (also referred to as MDC proteins, metalloprotease/disintegrin/cysteine-rich, or as ADAMs, a disintegrin and metalloprotease) are a family of membraneanchored glycoproteins that have roles in fertilization (16 -19), muscle fusion (20), release of tum...

Abstract. Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an ~84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 1251-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metaUoprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein.

Here we report the cloning and initial biochemical characterization of the mouse metalloprotease/disintegrin/cysteine-rich (MDC) protein meltrin ␤ and the analysis of the mRNA expression of four MDC genes (meltrin ␣, meltrin ␤, mdc9, and mdc15) in bone cells, including osteoclasts and osteoblasts. Like most other MDC proteins, the predicted meltrin ␤ protein consists of a signal sequence, prodomain, metalloprotease domain with a predicted catalytic site, disintegrin domain, cysteinerich region, epidermal growth factor repeat, transmembrane domain, and cytoplasmic domain with putative signaling motifs, such as potential SH3 ligand domains. Northern blot analysis indicates that meltrin ␤ is widely expressed, with the highest expression in bone, heart, and lung. RNase protection studies revealed expression of all four MDC genes analyzed here in osteoblasts, whereas only mdc9 and mdc15 mRNAs were detectable in osteoclast-like cells generated in vitro. Treatment of primary osteoblasts with 10 nM calcitriol increased meltrin ␤ expression more than 3-fold, and both meltrin ␣ and meltrin ␤ expression is apparently regulated in a differentiation-associated manner in a mouse osteoblastic cell line, MC3T3E1. Collectively, these results suggest that meltrin ␣ and meltrin ␤ may play a role in osteoblast differentiation and/or function but are not likely to be involved in osteoclast fusion.Proteins containing a metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC 1 proteins, also referred to as ADAMs (1)) are a family of membrane-anchored glycoproteins that are related to soluble snake venom metalloproteases and integrin ligands. MDC proteins have been implicated in a variety of important cellular processes, including sperm-egg and muscle cell membrane binding and fusion (2-6); in neuronal induction, lateral inhibition, and neuronal outgrowth in Drosophila (7-9); and in the release of the membrane-anchored cytokine tumor necrosis factor ␣ (10, 11). The finding that the tumor necrosis factor ␣ convertase is an MDC protein (10, 11) and that Drosophila Kuzbanian may be involved in the intracellular processing Notch (9, 12) raises the intriguing possibility that other membrane-anchored proteins are also processed and/or released by metalloprotease-disintegrin proteins (13). Because several MDC proteins contain potential cytoplasmic signaling motifs, including SH3 ligand domains (1, 14 -16), MDC proteins may also play a role in signaling events, or they may be regulated through interactions with cytoplasmic proteins. At present, over 20 genes encoding MDC proteins have been identified in different species, including Caenorhabditis elegans (17), Drosophila melanogaster (7,8), Xenopus laevis (18, 19), mice (1,6,14,20,21), guinea pigs (1,4,21,22), and humans (10,11,14,15,23). Although the function of many of these proteins remains to be determined, it is clear that different MDC proteins are capable of performing a variety of important tasks. To date, MDC proteins have been implicated in two distinct membrane...

Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic Xgtll expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by rinding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid selection of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of :3.6 and r2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.Myeloperoxidase (MPO), a critical microbicidal protein of mature polymorphonuclear neutrophils, is a hemoprotein composing 3-5% of the protein weight of these cells (1, 2). It appears in the primary azurophilic granules during the differentiation of the neutrophil (3, 4) and has been used as a marker for these granules (5). Cytochemical staining of MPO activity is used clinically to distinguish acute myeloid leukemia from acute lymphoid leukemia (6). Furthermore, approximately 1 in every 2000 individuals has been shown to be deficient in MPO (7), an apparently benign but poorly understood hereditary disorder of polymorphonuclear neutrophils.Although the function of MPO in the oxygen-dependent respiratory burst of polymorphonuclear neutrophils has been firmly established, relatively little is known about MPO biosynthesis, posttranslational modification, and genetic organization and expression. The exact structure of MPO remains a source of some controversy, and its amino acid sequence is not known.The appearance of MPO in the primary granules marks the transition from the myeloblast to the promyelocyte in the course of normal neutrophilic differentiation (3). Promyelocytes, however, are not found in the peripheral blood of normal individuals and are therefore difficult to study biochemically. The HL-60 cell line, derived from a patient with acute promyelocytic leukemia, contains virtually 100%o promyelocytes with azurophilic granules containing MPO (8).When HL-60 cells are cultured in the presence of compounds such as dimethyl sulfoxide (Me2SO) (8) or retinoic acid (9), these cells mature into metamyelocytes, neutrophili...

Acute promyelocytic leukemia (subtype M3) is characterized by malignant promyelocytes exhibiting an abundance of abnormally large or aberrant primary granules. Myeloperoxidase (MPO) activity of these azurophilic granules, as assessed by cytochemical staining, is unusually intense. In addition, M3 is universally associated with a chromosomal translocation, t(15;17)(q22;q11.2). In this report, the MPO gene was localized to human chromosome 17 (q12-q21), the region of the breakpoint on chromosome 17 in the t(15;17), by somatic cell hybrid analysis and in situ chromosomal hybridization. By means of MPO complementary DNA clones for in situ hybridization and Southern blot analysis, the effect of this specific translocation on the MPO gene was examined. In all cases of M3 examined, MPO is translocated to chromosome 15. Genomic blot analyses indicate rearrangement of MPO in leukemia cells of two of four cases examined. These findings suggest that MPO may be pivotal in the pathogenesis of acute promyelocytic leukemia.