Sialyllactose-mediated cell interaction during granulosa cell differentiation. Identification of its binding proteins.
The present study was designed to prove the carbohydrate-binding proteins interacting with cell surface sialyllactosylceramide (GM3, NeuAc alpha 2-->3Gal beta 1-->4Glc beta 1-->1'Cer), which is highly expressed during differentiation of rat ovarian granulosa cells. As a specific ligand for the sialyllactose (SL)-binding proteins on granulosa cells, we used a radioiodinated multivalent SL-linked albumin (Alb-(SL)17). The specific association of the ligand to the putative proteins on the intact cells was competitively inhibited by GM3 more effectively than other gangliosides, sialyllactotetraosylceramide, sialylneolactotetraosylceramide, and several glycoproteins with N-linked oligosaccharides. However, the proteins had no specificity for the side chain (N-acetyl or N-glycolyl forms) of sialic acid in GM3. Scatchard analysis of Alb-(SL)17 binding showed high (Kd = 6.4 x 10(-10)M) and low (Kd = 3.1 x 10(-8)M) affinity population of binding sites. By direct binding of 125I-Alb-(SL)17 to SL-binding proteins on Western blots, the putative proteins with molecular masses of 35, 18, and 14 kDa were detected. The interaction of the multivalent derivative with these binding proteins was differently modulated by Ca2+ and Mn2+. The SL-binding proteins occurred in immature granulosa cells and progressively decreased during differentiation, whereas their endogenous ligand GM3 increased. These results indicate that relatively low molecular weight SL-binding proteins exist on the surface of immature granulosa cells and that they may serve as receptor sites for newly synthesized GM3 during differentiation.
Hattori
M
M
Department of Cell Biology, Gunma University, Japan.
Horiuchi
R
R
Hosaka
K
K
Hayashi
H
H
Kojima
I
I
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002240:Carbohydrate Sequence; D002450:Cell Communication; D002454:Cell Differentiation; D002478:Cells, Cultured; D005260:Female; D005679:G(M3) Ganglioside / Q000096:biosynthesis; D006107:Granulosa Cells / Q000166:cytology*; D007457:Iodine Radioisotopes; D007785:Lactose / Q000031:analogs & derivatives* / Q000378:metabolism / Q000502:physiology; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D012709:Serum Albumin / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism / Q000502:physiology*
D005679:G(M3) Ganglioside; D007457:Iodine Radioisotopes; D012709:Serum Albumin; D012794:Sialic Acids; C000020:N-acetylneuraminoyllactose; D008345:Manganese; D007785:Lactose; D002118:Calcium