Carbohydrate removal fails to eliminate the heterogeneity of human prostatic acid phosphatase.
Human prostatic acid phosphatase is known to display considerable charge heterogeneity upon isoelectric focusing. The structural basis of this heterogeneity is not known, although it has been widely attributed to variations in the nature of the carbohydrate chains or to substituents on the carbohydrate chains of the glycoprotein. In this study, the role of the carbohydrate chains in the charge heterogeneity of the protein was examined. First, sialic acid residues were removed by treatment of the acid phosphatase with neuraminidase. The desialo enzyme was fractionated and purified by L-tartramic acid affinity chromatography. Then, after the protein oligosaccharide linkages were made accessible by the presence of NP-40 or by denaturing the protein, the protein was completely deglycosylated by endo-beta-N-acetylglucosaminidase F at pH 4.5 and 9.3. Two discrete intermediates were clearly resolved by SDS gel electrophoresis during the deglycosylation of the denatured protein at pH 9.3, indicating the existence of three sites of glycosylation on the protein. Peptide mixtures were obtained by digestion of carboxymethylated and citraconylated derivatives of the enzyme with trypsin and the glycopeptides were isolated. The amino acid compositions of the glycopeptides were consistent with the interpretation that there are a minimum of two sites of glycosylation on each peptide subunit of the enzyme. Isoelectric focusing experiments on the native, desialo, and denatured, deglycoso acid phosphatase showed that the heterogeneity of the protein is not eliminated either by desialylation or by deglycosylation. Thus, the electrophoretic heterogeneity of human prostatic acid phosphatase does not lie primarily in the oligosaccharide part of the glycoprotein or in altered conformational states of the protein, but in structural variations of the polypeptide itself. The heterogeneity may be due to variations at the C-terminus, partial deamidation, phosphorylation, sulfation or other posttranslational modifications of the protein chain.
Morris
M F
MF
Department of Chemistry, Purdue University, West Lafayette, IN 47907.
Waheed
A
A
Risley
J M
JM
Van Etten
R L
RL
D000117:Acetylglucosamine / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000378:metabolism; D000135:Acid Phosphatase / Q000302:isolation & purification / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates; D002846:Chromatography, Affinity; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011467:Prostate / Q000201:enzymology*; D012794:Sialic Acids / Q000378:metabolism; D013329:Structure-Activity Relationship; D014357:Trypsin
D000596:Amino Acids; D002241:Carbohydrates; D010446:Peptide Fragments; D012794:Sialic Acids; D000135:Acid Phosphatase; D009439:Neuraminidase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin; D019158:N-Acetylneuraminic Acid; D000117:Acetylglucosamine