Purification and characterization of a glycosylated proline-rich protein from human parotid saliva.
1. A glycosylated proline-rich protein (GPRP) was purified to homogeneity by subjecting parotid saliva to immunoaffinity, cation exchange, affinity and hydrophobic chromatography. 2. The purified GPRP had a molecular weight of 78 kDa as analyzed by SDS-PAGE. 3. The amino acid analysis revealed a preponderance of proline, glycine and glutamic acid/glutamine, which accounted for 77% of the total amino acids. 4. Cysteine, tyrosine or phenylalanine residues were not detected. 5. The glycoprotein contained 34% neutral sugars and the oligosaccharides were rich in mannose and N-acetylglucosamine, indicating that N-linked oligosaccharides were the predominant type of oligosaccharides in the molecule. 6. These observations were confirmed by treatment of the purified glycoprotein with specific N-glycosidase which removed the N-linked oligosaccharides leaving a core protein with an apparent molecular weight of 51 kDa. 7. The isoelectric point of GPRP was approx 7.0 and the molecule was not affected by reduction with 2-mercaptoethanol, indicating that no disulfide linkages were present. 8. The GPRP bound to hydroxyapatite and this binding could be partially inhibited by preincubation of the hydroxyapatite with parotid or submandibular saliva. 9. The purified GPRP also bound to a protein with an apparent molecular weight of 95 kDa present in submandibular saliva.
Oho
T
T
Department of Preventive Dentistry, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
Rahemtulla
F
F
Månsson-Rahemtulla
B
B
Hjerpe
A
A
D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D002241:Carbohydrates / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006801:Humans; D010306:Parotid Gland / Q000737:chemistry*; D011392:Proline / Q000032:analysis*; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000302:isolation & purification*
D000596:Amino Acids; D002241:Carbohydrates; D012471:Salivary Proteins and Peptides; D011392:Proline