Splice-site genetic polymorphism of the human kallikrein 12 (KLK12) gene correlates with no substantial expression of KLK12 protein having serine protease activity.
The human kallikrein 12 (KLK12) gene is a new member of the KLK gene family, some members of which are implicated in the initiation and progression of cancer. In this study, we examined 50 non-cancerous tissues from Japanese patients with primary gastric cancer to determine the presence of genetic polymorphisms in the KLK12 gene using polymerase chain reaction (PCR)-single-strand conformation polymorphism and sequencing. Four different types of genetic polymorphisms were identified: one at a splice-donor site of intron 4 (c.457+2T>C), two in exon 6 (c.618_619delTG:p.Cys206fsX72 and c.735G>A:p.Met245Ile), and one in intron 3. The c.457+2T>C polymorphism was observed at a high frequency (allele frequency:0.63), compared to the frequencies of the two polymorphisms in exon 6 (allele frequency:0.01). Reverse transcriptase (RT)-PCR and Western blot analyses revealed that the c.457+2T>C polymorphism was associated with a splicing abnormality and that the expression of the human KLK12 protein (hK12), corresponding to the putative serine protease, was absent in individuals with a c.457+2C/C genotype but not in individuals with the T/T or T/C genotypes. We also found that recombinant His6-tagged hK12 has activity that cleaves chromogenic substrate (H-D-Pro-L-Phe-L-Arg-p-nitroaniline dihydrochloride), that is, serine protease activity. These results indicate that individuals with the c.457+2C/C genotype have no substantial expression of hK12 serine protease.
First Department of Pathology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan. hsugimur@hama-med.ac.jp
Kazuya
K
Shinmura
Hong
H
Tao
Hidetaka
H
Yamada
Hideki
H
Kataoka
Ravshanov
R
Sanjar
Jiandong
J
Wang
Kimio
K
Yoshimura
Haruhiko
H
Sugimura
D002277:Carcinoma / Q000201:enzymology / Q000453:epidemiology / Q000235:genetics; D002460:Cell Line; D002863:Chromogenic Compounds / Q000378:metabolism; D004790:Enzyme Induction; D005091:Exons / Q000235:genetics; D016368:Frameshift Mutation; D005753:Gastric Mucosa / Q000201:enzymology; D005838:Genotype; D006801:Humans; D007438:Introns / Q000235:genetics; D007564:Japan / Q000453:epidemiology; D007610:Kallikreins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics*; D015810:Linkage Disequilibrium; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D022821:RNA Splice Sites / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013274:Stomach Neoplasms / Q000201:enzymology / Q000453:epidemiology / Q000235:genetics; D014162:Transfection
D002863:Chromogenic Compounds; D022821:RNA Splice Sites; D012333:RNA, Messenger; C497170:KLK12 protein, human; D007610:Kallikreins