title: Characterization of the Glycosylation Site of Human PSA Prompted by Missense Mutation using LC-MS/MS.
abstract: Prostate specific antigen (PSA) is currently used as a diagnostic biomarker for prostate cancer. It is a glycoprotein possessing a single glycosylation site at N69. During our previous study of PSA N69 glycosylation, additional glycopeptides were observed in the PSA sample that were not previously reported and did not match glycopeptides of impure glycoproteins existing in the sample. This extra glycosylation site of PSA is associated with a mutation in KLK3 genes. Among single nucleotide polymorphisms (SNPs) of KLKs families, the rs61752561 in KLK3 genes is an unusual missense mutation resulting in the conversion of D102 to N in PSA amino acid sequence. Accordingly, a new N-linked glycosylation site is created with an N102MS motif. Here we report the first qualitative and quantitative glycoproteomic study of PSA N102 glycosylation site by LC-MS/MS. We successfully applied tandem MS to verify the amino acid sequence possessing N102 glycosylation site and associated glycoforms of PSA samples acquired from different suppliers. Among the three PSA samples, HexNAc2Hex5 was the predominant glycoform at N102, while HexNAc4Hex5Fuc1NeuAc1 or HexNAc4Hex5Fuc1NeuAc2 was the primary glycoforms at N69. D102 is the first amino acid of "kallikrein loop", which is close to a zinc-binding site and catalytic triad. The different glycosylation of N102 relative to N69 might be influenced by the close vicinity of N102 to these functional sites and steric hindrance.
authors.forename: Ehwang
authors.initials: E
authors.lastname: Song
authors.forename: Yunli
authors.initials: Y
authors.lastname: Hu
authors.affiliation: ∥Department of Biotechnology, Alexandria University, 163-Horreya Avenue, El-Shatby 21526, Alexandria, Egypt.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Hussein
authors.forename: Chuan-Yih
authors.initials: CY
authors.lastname: Yu
authors.forename: Haixu
authors.initials: H
authors.lastname: Tang
authors.forename: Yehia
authors.initials: Y
authors.lastname: Mechref
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D020641:Polymorphism, Single Nucleotide; D017430:Prostate-Specific Antigen / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
chemical_list: D017430:Prostate-Specific Antigen
keywords: D102N; LC−MS/MS; N-linked glycosylation; PSA; glycopeptide; glycoproteomics; missense mutation; prostate specific antigen; rs61752561