Graduate School of Technology, Niigata Institute of Technology, Niigata 945-1195, Japan.
Graduate School of Technology, Niigata Institute of Technology, Niigata 945-1195, Japan.
Arch Oral Biol. 2018 Apr;88:10-18. doi: 10.1016/j.archoralbio.2018.01.006. Epub 2018 Jan 10.
The NCBI gene database and human-transcriptome database for alternative splicing were used to determine the expression of mRNAs for P-B (SMR3B) and variant form of P-B. The translational product from the former mRNA was identified as the protein named P-B, whereas that from the latter has not yet been elucidated. In the present study, we investigated the expression of P-B and its variant form at the protein level.
To identify the variant protein of P-B, (1) cationic proteins with a higher isoelectric point in human pooled whole saliva were purified by a two dimensional liquid chromatography; (2) the peptide fragments generated from the in-solution of all proteins digested with trypsin separated and analyzed by MALDI-TOF-MS; and (3) the presence or absence of P-B in individual saliva was examined by 15% SDS-PAGE.
The peptide sequences (IPPPYSCTPNMNNCSR, CHHHHKRHHYPCNYCFCYPK, RHHYPCNYCFCYPK and HHYPCNYCFCYPK) present in the variant protein of P-B were identified. The peptide sequence (GPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR) in P-B (or the variant) and sequence (IPPPPPAPYGPGIFPPPPPQP) in P-B were identified. The sum of the sequences identified indicated a 91.23% sequence identity for P-B and 79.76% for the variant. There were cases in which P-B existed in individual saliva, but there were cases in which it did not exist in individual saliva.
The variant protein is produced by excising a non-canonical intron (CC-AC pair) from the 3'-noncoding sequence of the PBII gene. Both P-B and the variant are subject to proteolysis in the oral cavity.
利用 NCBI 基因数据库和人类转录组数据库中选择性剪接的信息，确定 P-B（SMR3B）及其变体形式的 mRNA 的表达情况。前一种 mRNA 的翻译产物被鉴定为名为 P-B 的蛋白质，而后者的翻译产物尚未阐明。在本研究中，我们在蛋白质水平上研究了 P-B 及其变体形式的表达情况。
为了鉴定 P-B 的变体蛋白，（1）通过二维液相色谱法从人混合全唾液中分离具有较高等电点的阳离子蛋白；（2）通过 MALDI-TOF-MS 对经胰蛋白酶消化后的所有蛋白质溶液中生成的肽片段进行分离和分析；（3）通过 15%SDS-PAGE 检测个体唾液中 P-B 的存在情况。
鉴定出 P-B 变体蛋白中存在的肽序列（IPPPYSCTPNMNNCSR、CHHHHKRHHYPCNYCFCYPK、RHHYPCNYCFCYPK 和 HHHYPCNYCFCYPK）。鉴定出 P-B（或变体）中的肽序列（GPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR）和 P-B 中的序列（IPPPPPAPYGPGIFPPPPPQP）。鉴定出的序列总和表明 P-B 的序列同一性为 91.23%，变体的序列同一性为 79.76%。在个体唾液中存在 P-B 的情况，也存在不存在 P-B 的情况。
变体蛋白是通过从 PBII 基因的 3'-非编码序列中切除一个非典型内含子（CC-AC 对）而产生的。P-B 和变体都受到口腔内蛋白水解的影响。
