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PMID: 7717985 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Expression of wild-type and mutated rabbit osteopontin in Escherichia coli, and their effects on adhesion and migration of P388D1 cells.

The Biochemical journal ·Vol. 307 ( Pt 1) ·1995-04-01 ·Pages 257-65

Nasu K, Ishida T, Setoguchi M, Higuchi Y, Akizuki S, Yamamoto S

Abstract

Recombinant wild-type rabbit osteopontin (rOP) and the protein with an aspartate-to-glutamate transposition induced by a point mutation in the rabbit OP cDNA within the Gly-Arg-Gly-Asp-Ser (GRGDS) sequence were expressed in Escherichia coli and purified to homogeneity. P388D1 cells bound rOP in a saturable manner. rOP induced adhesion and haptotaxis of P388D1 cells, whereas mutated rabbit OP (rOPmut) did not. Anti-rOP IgG F(ab')2 and synthetic GRGDS peptide inhibited rOP-mediated adhesion and haptotaxis of P388D1 cells. Fibronectin (FN)-mediated adhesion of P388D1 cells was markedly inhibited in the presence of fluid-phase rOP. Adhesion of P388D1 cells to rOP was significantly inhibited by anti-[alpha-subunits of VLA4 (alpha 4) and VLA5 (alpha 5)] monoclonal antibodies (mAbs), but not by anti-[alpha-subunit of vitronectin (VN) receptor (alpha V) or Mac-1 (alpha M)] mAb. Adhesion of P388D1 cells to FN and VN was significantly inhibited by anti-alpha V mAb but not anti-alpha 4, -alpha 5 or -alpha M mAb. Haptotaxis of P388D1 cells to rOP was significantly inhibited by anti-alpha V mAb, but not by anti-alpha 4, -alpha 5 and alpha M mAbs, whereas that to FN showed no inhibition with all three mAbs. Haptotaxis of P388D1 cells to VN was significantly inhibited by anti-alpha 5 and -alpha V mAbs but not by anti-alpha 4 and -alpha M mAbs. Similar features of inhibition of adhesion and haptotaxis of P388D1 cells to human OP were observed by mAbs. rOP had no chemotactic effect on P388D1 cells. Significant polymorphonuclear leucocyte migration was observed 3-12 h after intradermal injection of rOP into rabbits.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Cell Adhesion/drug effects Cell Movement/drug effects DNA, Complementary/genetics Escherichia coli/genetics Fibronectins Immune Sera Leukemia P388/pathology Leukocytes/drug effects Mice Molecular Sequence Data Mutagenesis, Site-Directed Oligopeptides/physiology Osteopontin Protein Binding Rabbits/genetics,immunology Recombinant Fusion Proteins/biosynthesis,genetics,immunology,pharmacology Sequence Alignment Sialoglycoproteins/biosynthesis,genetics,immunology,pharmacology Tumor Cells, Cultured/drug effects
Chemicals
DNA, Complementary Fibronectins Immune Sera Oligopeptides Recombinant Fusion Proteins SPP1 protein, human Sialoglycoproteins Spp1 protein, mouse Osteopontin glycyl-arginyl-glycyl-aspartyl-serine
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Nasu K
Department of Pathology, Oita Medical University, Japan.
Ishida T
Setoguchi M
Higuchi Y
Akizuki S
Yamamoto S
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj3070257
Published
1995-04-01
Pages
257-65
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1136771
Subset
IM
Databases
GENBANK
D16544
External Links
PubMed source DOI: 10.1042/bj3070257 PMC Full Text View journal details
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