The tumor-associated antigen 90K/Mac-2-binding protein secreted by human colon carcinoma cells enhances extracellular levels of promatrilysin and is a novel substrate of matrix metalloproteinases-2, -7 (matrilysin) and -9: Implications of proteolytic cleavage |
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Authors: | Tricia A Ulmer Vicki Keeler Sabine André Hans-Joachim Gabius Lambert Loh Suzanne Laferté |
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Institution: | 1. Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Room A3, Saskatoon, Saskatchewan, Canada S7N 5E5;2. Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, Veterinärstr. 13, D-80539, Munich, Germany |
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Abstract: | BackgroundThe tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein is expressed at elevated level in cancerous tissues and associated with poor prognosis. Since TAA90K has been implicated in the restructuring of the extracellular matrix, we examined the functional relationship between colon cancer cell-derived TAA90K and the matrix metalloproteinase (MMP) promatrilysin (proMMP-7), and also tested whether TAA90K is a novel substrate for MMPs-2, -7 and -9.MethodsThe effect of TAA90K on proMMP-7 levels in HT-29 conditioned media was quantified by enzyme-linked immunosorbent assays. Binding of TAA90K to MMPs, extracellular matrix proteins and galectin-3 was measured by solid-phase binding assays. Proteolytic cleavage of TAA90K by MMPs was documented by SDS-PAGE and protein sequencing analysis.ResultsTAA90K enhanced extracellular levels of proMMP-7 in HT-29 cells. In addition, TAA90K was cleaved by MMPs-2, -7 and -9. MMP-7-mediated cleavage of TAA90K did not affect its binding to MMP-7, laminin-1, collagen IV and galectin-3 but reduced its interaction with fibronectin and laminin-10, and lowered the levels of proMMP-7 in the HT-29 medium.ConclusionTAA90K is a novel substrate for MMPs-2, -7 and -9 and modulates proMMP-7 levels in colon cancer cells.General significanceProteolytic cleavage of TAA90K may have functional implications in colon cancer. |
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Keywords: | APMA p-aminophenylmercuric acetate BSA bovine serum albumin CyCAP cyclophilin C-associated protein DMEM Dulbecco's minimum essential medium ECM extracellular matrix EDTA ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay FBS fetal bovine serum HPLC high-performance liquid chromatography IgG immunoglobulin G IL-1β interleukin-1β IL-6 interleukin-6 MEM minimal essential medium MMP matrix metalloproteinase PBS phosphate buffered saline PBST PBS/04% Tween 20 SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis TAA90K tumor-associated antigen 90K |
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