Biochemical characterization of integral membrane heparan sulfate proteoglycans in Sertoli cells from immature rat testis.
(35)S-Radiolabeled cultured Sertoli cells from immature rat testis were extracted with detergent and the different proteoheparan sulfate (HSPG) forms of the extract were discriminated and quantified on the basis of their high anionic charge, hydrodynamic size, lipophilic properties, susceptibility to trypsin and phosphatidylinositol phospholipase C (PI-PLC). Trypsin released 50% of total cellular HSPG corresponding to 80% of total hydrophobic HSPG. Trypsin-accessible HSPG were presumed to be integral membrane species. Trypsin-resistant HSPG, probably intracellular, distributed into non-lipophilic (37.5%) and lipophilic (12.5%) populations. Biochemical analysis of PG copurified with plasma membrane confirmed the existence of hydrophobic HSPG integrated into this structure. Among hydrophobic HSPG accessible to trypsin, 35% were PI-PLC released and radiolabeled by [(3)H]inositol indicating that about one third of integral membrane HSPG were intercalated into the plasma membrane through a phosphatidylinositol anchor (glypican type). PI-PLC-resistant forms represented HSPG inserted into the membrane through a hydrophobic segment of the core protein (syndecan type). No lipophilic PG was present in other cell compartments (culture medium, cell periphery, extracellular matrix). (125)I-Iodinated hydrophobic HSPG were deglycanated and submitted to SDS-polyacrylamide gel electrophoresis. In the glypican family, a core protein (64--65 kDa) was detected, whereas in the syndecan family, bands of 60 and 68 kDa were observed which may correspond to self-association of different core proteins. In Sertoli cell, specific functional attributes of different integral membrane HSPG forms remain to be investigated.
Brucato
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Laboratoire de Biochimie, UPRES EA 2608, I.R.B.A., Université de Caen, Esplanade de la Paix, F-14032 Caen Cedex, France. biochimie@ibba.unicaen.fr
Fagnen
G
G
Villers
C
C
Bonnamy
P J
PJ
Langris
M
M
Bocquet
J
J
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D017261:Glycosylphosphatidylinositols / Q000032:analysis; D019812:Heparan Sulfate Proteoglycans / Q000032:analysis*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012708:Sertoli Cells / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin; D010738:Type C Phospholipases
D017261:Glycosylphosphatidylinositols; D019812:Heparan Sulfate Proteoglycans; D010738:Type C Phospholipases; D014357:Trypsin