Kallikrein 4 is a secreted protein.
Kallikrein 4 is a secreted protein.
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激肽释放酶 4 是一种分泌蛋白。
DOI：
10.1158/0008-5472.can-04-1660
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发表时间：
2004
期刊：
影响因子：
11.2
通讯作者：
Bartlett,JohnD
中科院分区：
文献类型：
--
作者：
Simmer,JamesP;Bartlett,JohnD
To the editor: In the April 1, 2004 issue of Cancer Research, Xi et al.(1) reported that kallikrein 4 (KLK4) is predominantly a nuclear protein. They state that the KLK4 gene has only four exons when actually it has five (the first exon is noncoding; ref. 2), and they state that the first coding exon is not physiologically significant and is not present in the vast majority of KLK4 transcripts. We would like to raise several concerns about the experimental evidence used to support these conclusions.The authors performed routine reverse transcription-PCR reactions that paired a common 3-PCR primer with one of six different 5-primers. Using total RNA from LNCaP cells as substrate, the authors discovered that the 5-primers annealing to the first coding exon did not amplify KLK4 mRNA as efficiently as 5-primers specific for the second coding exon. From this, they concluded that:“The putative first (coding) exon of KLK4, if present is at extremely insignificant low levels compared with downstream sequences.” No other analyses of the KLK4 transcripts were performed. Perhaps nicking of the mRNA during isolation and/or failure of the reverse transcriptase to extend to the 5-end of the KLK4 transcripts can best explain these results. On the basis of their reverse transcription-PCR results, the authors’ appear to demonstrate that coding exon 2 is at the 5-end of their KLK4 mRNA. This is not possible because the 5-end of coding exon 2 contains a consensus splice junction and not the required transcription initiation site. However, even if we assume that coding exon 2 does contain a valid transcription initiation site, the first ATG (Met50) is located midway through the exon (Fig. 1). Initiation at the Met50 codon deletes the signal peptide (Met1-Val24), propeptide (S25-Q30), and the NH2 terminus of the active enzyme (I32-V49). The KLK4 antibody the authors used to demonstrate nuclear localization was raised against the NH2-terminal peptide QIINGEDCSPHSQPW (Q31-W45). This antibody would not have detected a protein translated from transcripts starting with coding exon 2. This is a major inconsistency. The authors used this antibody for their Western blot analyses to identify a protein of 45 kDa, but the immunopositive protein was not characterized additionally. Previously, a rabbit polyclonal antibody raised against recombinant human KLK4 demonstrated that human KLK4 (fractionated from prostate cancer cytosolic extracts) had a molecular mass of 30 kDa (3). Immunohistochemistry with this antibody did not detect KLK4 in the nucleus. Cross-reaction of the anti-peptide KLK4 antibody with an unidentified nuclear protein appears the most reasonable interpretation of the authors’ data.
