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ABSTRACT: Endomucin is an endothelial sialomucin that was recently identified with the help of monoclonal antibodies raised against mouse endothelial cells. Cloning of human endomucin allowed us to generate monoclonal antibodies against soluble recombinant forms of human endomucin. In this study, we investigated the expression of this novel molecule in human skin under different conditions, using the monoclonal antibodies. In normal human skin, endomucin was detected for the monoclonal antibody L6H10 by immunoblotting, and immunohistologic analysis of wax-embedded sections revealed that this glycoprotein is expressed on capillaries, venules, and lymphatic vessels. Interestingly, staining of arterial endothelium was either weak or focal using the monoclonal antibodies against endomucin. In situ hybridization of normal human skin confirmed the expression pattern on the messenger RNA level obtained above. We further analyzed the expression of endomucin in skin biopsy specimens from patients with inflammatory skin diseases, such as atopic dermatitis, psoriasis, lichen planus, cutaneous lupus erythematosus, and T cell lymphoma as well as with vascular skin tumors, such as hemangioma, pyogenic granuloma, angiolipoma, Kaposi's sarcoma, and angiosarcoma. We found endomucin expressed on the endothelium of each tissue, concluding that this novel molecule is a new endothelial-specific marker in the study of normal and diseased human skin.
Journal of Investigative Dermatology 01/2003; 119(6):1388-93. · 6.31 Impact Factor
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Irene Yiallouros,
Reinhild Kappelhoff,
Oliver Schilling,
Frank Wegmann,
Mike W Helms,
Astrid Auge, Gertrud Brachtendorf,
Eva Grosse Berkhoff,
Bernd Beermann,
Hans Jürgen Hinz,
Simone König,
Jasna Peter-Katalinic,
Walter Stöcker
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ABSTRACT: Astacin (EC 3.4.24.21) is a prototype for the astacin family and for the metzincin superfamily of zinc peptidases, which comprise membrane-bound and secreted enzymes involved in extracellular proteolysis during tissue development and remodelling. Generally, metzincins are translated as pro-enzymes (zymogens), which are activated by removal of an N-terminal pro-peptide. In astacin, however, the mode of zymogen activation has been obscured, since the pro-form does not accumulate in vivo. Here we report the detection of pro-astacin in midgut glands of brefeldin A-treated crayfish (Astacus astacus) by immunoprecipitation and mass spectrometry. We demonstrate that the pro-peptide is able to shield the active site of mature astacin as a transient inhibitor, which is degraded slowly. In vitro studies with recombinant pro-astacin in the absence of another protease reveal a potential of auto-proteolytic activation. The initial cleavage in this autoactivation appears to be an intramolecular event. This is supported by the fact that the mutant E93A-pro-astacin is incapable of autoactivation, and completely resistant to cleavage by mature astacin. However, this mutant is cleaved by Astacus trypsin within the pro-peptide. This probably reflects the in vivo situation, where Astacus trypsin and astacin work together during pro-astacin activation. In a first step, trypsin produces amino-terminally truncated pro-astacin derivatives. These are trimmed subsequently by each other and by astacin to yield the mature amino terminus, which forms a salt-bridge with Glu103 in the active site. The disruption of this salt-bridge in the mutants E103A and E103Q results in extremely heat labile proteins, whose catalytic activities are not altered drastically, however. This supports a concept according to which the linkage of Glu103 to the precisely trimmed amino terminus is a crucial structural prerequisite throughout the astacin family.
Journal of Molecular Biology 12/2002; 324(2):237-46. · 4.00 Impact Factor
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Ines Nasdala,
Karen Wolburg-Buchholz,
Hartwig Wolburg,
Annegret Kuhn,
Klaus Ebnet, Gertrud Brachtendorf,
Ulrike Samulowitz,
Bernhard Kuster,
Britta Engelhardt,
Dietmar Vestweber,
Stefan Butz
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ABSTRACT: Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.
Journal of Biological Chemistry 06/2002; 277(18):16294-303. · 4.77 Impact Factor
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ABSTRACT: Endomucin is a typical sialomucin that we recently identified on the surface of mouse endothelial cells and on putative hematopoetic clusters of the dorsal aorta in the embryo. We have generated a panel of monoclonal antibodies (mAbs) against the extracellular part of human endomucin and polyclonal antibodies against the cytoplasmic part. Using immunohistochemistry endomucin was specifically detected on endothelial cells of blood and lymphatic vessels of all analyzed human tissues. In addition, the polyclonal antibodies stained the epithelium of the epidermis as well as epithelial and myoepithelial cells of the eccrine and apocrine glands in the skin. This nonendothelial staining could only be seen with a subset of mAbs if the staining procedure was amplified. Although high endothelial venules (HEVs) were not significantly stained with mAbs against endomucin, the polyclonal antibodies clearly detected endomucin on HEVs in lymphatic organs of the mouse and human, suggesting HEV-specific glycosylation affecting recognition by the mAbs. Indeed, endomucin isolated from human and mouse lymphoid organs carried the MECA-79 epitope that defines a set of L-selectin ligands on HEVs called peripheral node addressins. We conclude that human and mouse endomucin are endothelial sialomucins with the potential to function as L-selectin ligands.
American Journal Of Pathology 06/2002; 160(5):1669-81. · 4.89 Impact Factor
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ABSTRACT: Endomucin is a recently identified sialomucin that is specifically expressed on endothelium of the adult mouse. Here, we have analysed the expression of endomucin during development of the vascular system by immunohistochemistry by using three monoclonal antibodies (mAb). We demonstrate that two of the mAb, V.5C7 and V.1A7, recognize epitopes on the nonglycosylated protein, because they recognize the antigen when it is synthesized as a bacterial fusion protein and when it is in vitro translated in a membrane-free reticulocyte lysate. During in vitro differentiation of embryonic stem cells to endothelial cells, endomucin is expressed at day 6 after onset of differentiation, 1 day later than PECAM-1. During differentiation of the mouse embryo, endomucin is first detected at E8.0 in all embryonic blood vessels detectable at this stage but is absent in blood islands of the yolk sac. Analysing the paraaortic-splanchnopleura (P-SP) region and the aorta-gonad-mesonephros (AGM) region as sites of intraembryonic hematopoiesis, we found that endothelium of the dorsal aorta is brightly positive for endomucin at E8.5–9.0 and at E11.5. At later stages and in the adult aorta, endothelial staining is strongly reduced and confined to focal areas. Cell clusters associated with the luminal surface of the endothelium of the dorsal aorta could be stained for endomucin and for CD34. At a later stage (E15.5) single leukocytes in the lumen of large venules were stained for endomucin. We conclude that endomucin is an early endothelial-specific antigen that is also expressed on putative hematopoietic progenitor cells. © 2001 Wiley-Liss, Inc.
Developmental Dynamics 10/2001; 222(3):410 - 419. · 2.54 Impact Factor
