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Characterization of the Glycosylation Site of Human PSA Prompted by Missense Mutation using LC–MS/MS
journal contribution
posted on 2015-07-02, 00:00 authored by Ehwang Song, Yunli Hu, Ahmed Hussein, Chuan-Yih Yu, Haixu Tang, Yehia MechrefProstate
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.