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Proteoglycan production by human glomerular visceral epithelial cells and mesangial cells in vitro.
- Source :
-
The Biochemical journal [Biochem J] 1995 May 01; Vol. 307 ( Pt 3), pp. 759-68. - Publication Year :
- 1995
-
Abstract
- Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G-50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement-membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular-basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular-basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement-membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perlecan mRNA, demonstrated using reverse transcriptase PCR, was different in the two cell types. We conclude that glomerular visceral epithelial cells and mesangial cells have characteristic patterns of proteoglycan production. Glomerular visceral epithelial cells produced a hybrid proteoglycan containing CS and HS independently attached to its core protein.(ABSTRACT TRUNCATED AT 250 WORDS)
- Subjects :
- Adolescent
Adult
Aged
Base Sequence
Cells, Cultured
Child
Child, Preschool
Chondroitin Sulfate Proteoglycans analysis
Chondroitin Sulfate Proteoglycans biosynthesis
Chromatography, High Pressure Liquid
DNA, Complementary biosynthesis
DNA, Complementary genetics
Enzyme-Linked Immunosorbent Assay
Epithelium metabolism
Glucosamine metabolism
Heparan Sulfate Proteoglycans
Heparitin Sulfate biosynthesis
Heparitin Sulfate genetics
Humans
Infant
Kidney Glomerulus cytology
Leucine metabolism
Middle Aged
Molecular Sequence Data
Polymerase Chain Reaction
Proteoglycans genetics
RNA genetics
RNA isolation & purification
RNA metabolism
RNA, Messenger analysis
RNA, Messenger genetics
Sulfates metabolism
Sulfur Radioisotopes
Transcription, Genetic
Tritium
Glomerular Mesangium metabolism
Kidney Glomerulus metabolism
Proteoglycans biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 0264-6021
- Volume :
- 307 ( Pt 3)
- Database :
- MEDLINE
- Journal :
- The Biochemical journal
- Publication Type :
- Academic Journal
- Accession number :
- 7537959
- Full Text :
- https://doi.org/10.1042/bj3070759