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Title: Molecular cloning and expression study of MADM: A metalloprotease with a potential integrin-binding domain.
Authors: Lu, Xiaohong.
First Published: 1996
Award date: 1996
Abstract: Mammalian disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. From cDNA libraries of human U937 lymphoma cells and human (adult) brain temporal cortex, a novel integral membrane metalloprotease named MADM (Mammalian Disintegrin-Metalloprotease) was cloned by probing with the bovine cDNA homologue. MADM contains several distinct protein domains: preproprotein, metalloprotease, disintegrin (potential integrin-binding domain), cysteine- rich, transmembrane and cytoplasmic tail. The deduced bovine and human mature protein sequences are 97 % identical while a unique insertion domain (52 residues) was found in the prodomain of human MADM. Northern blotting analysis revealed that two different sizes (4.5 kb and 3.2 kb) of MADM mRNA transcripts were expressed at low levels in a variety of transformed human cell lines. Western blotting of various mammalian cells with an antiserum to a peptide in the disintegrin domain detected a glycoprotein of 62 kDa. Immunolocalisation of MADM showed that it is expressed mainly in an intracellular compartment but was also expressed on the surface of a small percentage of some human cell lines. Three approaches were tried to express recombinant MADM in eukaryotic cells in order to study putative interactions of MADM with cognate integrins and pro tease substrates. Recombinant bovine MADM was expressed transiently in monkey COS cells but most of the expressed proteins were the precursor of MADM and the expression levels of recombinant mature protein were not sufficiently increased over endogenous level to allow functional studies. In a second approach, bovine MADM was expressed stably in murine erythroleukaemia (MEL) cells. Nine individual stably transfected MEL cell clones appeared to express recombinant bovine MADM but the lack of reactivity of this protein with the monoclonal antibody CG4 suggested that some of its disulphide bonds had misformed. The third approach was to achieve high expression of MADM in a baculovirus/insect cell system. A recombinant baculovirus containing a soluble disintegrin-cysteine rich domain polypeptide of human MADM (hDC) was successfully generated and conditions established to isolate high levels of recombinant hDC protein either from cell lysates or from the conditioned medium. Recombinant hDC protein would provide means to investigate putative interactions of MADM with integrins or other proteins and produce antibody reactive with native human MADM.
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Theses, College of Medicine, Biological Sciences and Psychology
Leicester Theses

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