Purification and characterization of a sulfated glycoprotein secreted by Sertoli cells

• Published in: Biochemistry (Easton) Vol. 25; no. 23; pp. 7265 - 7270
• Main Authors: Griswold, Michael D, Roberts, Ken, Bishop, Paul
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 18.11.1986
• Subjects: Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Carbohydrates - analysis; Cells, Cultured; Chromatography, Gel; Chromatography, High Pressure Liquid; Clusterin; Fundamental and applied biological sciences. Psychology; Glycoproteins; Glycoproteins - isolation & purification; Glycoproteins - metabolism; Male; Molecular Chaperones; Molecular Weight; Proteins; Rats; Sertoli Cells - metabolism; Sertoli cell; Vertebrata; Mammalia; Purification; Rat; Rodentia; Glycoproteins; Sulfatation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00371a003

Sulfated glycoprotein 2 (SGP-2), the major secretion product of Sertoli cells, was purified from cell culture medium by reverse-phase high-performance liquid chromatography. The native protein consists of disulfide-linked monomers of 41,000 and 29,000 daltons which have a strong tendency to associate into multimers. The purified SGP-2 was subjected to amino acid analysis and contained high levels of Asx (11.1%), Glx (15.1%), and leucine (11.5%). The oligosaccharides on the purified SGP-2 were analyzed to determine the monosaccharide compositions and the molecular weights of the intact carbohydrate moieties. SGP-2 was shown to be 23.7% carbohydrate and consisted of 1% fucose, 3.5% mannose, 4.1% galactose, 7.1% N-acetylglucosamine, and 8.0% N-acetylneuraminic acid. No N-acetylgalactosamine was detected. When the SGP-2 was digested with proteases, the intact oligosaccharides were chromatographed over a Bio-Gel P-6 column and found to elute in a single symmetrical peak of approximately 3,300 g/mol. On the basis of these results, the oligosaccharides on SGP-2 were proposed to consist of triantennary chains similar to those found on fetuin. When the 35SO4(2-)-labeled SGP-2 was digested with Pronase, the free amino acids could be separated by chromatography from the oligosaccharide. The 35SO4(2-) was shown to be associated with the oligosaccharide portion of SGP-2.