Kallikrein 4 is a secreted protein.

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 3Ј-PCR primer with one of six different 5Ј-primers. Using total RNA from LNCaP cells as substrate, the authors discovered that the 5Ј-primers annealing to the first coding exon did not amplify KLK4 mRNA as efficiently as 5Ј-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 5Ј-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 5Ј-end of their KLK4 mRNA. This is not possible because the 5Ј-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 (Met 50) is located midway through the exon (Fig. 1). Initiation at the Met 50 codon deletes the signal peptide (Met 1-Val 24), propeptide (S 25-Q 30), and the NH 2 terminus of the active enzyme (I 32-V 49). The KLK4 antibody the authors used to demonstrate nuclear localization was raised against the NH 2-terminal peptide QIINGEDCSPHSQPW (Q 31-W 45). This an-tibody 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 an-tibody raised against recombinant human KLK4 demonstrated that human KLK4 (fractionated from prostate cancer cytosolic extracts) had a molecular mass of ϳ30 kDa (3). Immunohistochemistry with this antibody did not detect KLK4 in the nucleus. Cross-reaction …