A consolidated catalogue and graphical annotation of dbSNP polymorphisms in the human tissue kallikrein (KLK) locus.

The human tissue kallikreins, 15 secreted serine proteases, may play diverse roles in pathophysiology. The National Center for Biotechnology Information's dbSNP was mined for polymorphisms located within the kallikrein (KLK) locus using custom-designed "ParSNPs" and "LocusAnnotator" software tools. Using "ParSNPs", a filterable catalogue of 1856 KLK polymorphisms (1023 validated) was generated. "LocusAnnotator" was used to annotate the KLK locus sequence with gene and polymorphism features. A second locus was examined to validate the use of both programs on a non-kallikrein locus. This report may assist in the informed selection of KLK polymorphisms for future association and biochemical studies in relation to disease. Furthermore, "ParSNPs" and "LocusAnnotator" are available at no cost from our website (www.acdcLab.org/annotations) to examine other loci.

In silico identification and Bayesian phylogenetic analysis of multiple new mammalian kallikrein gene families.

Kallikrein gene families have been identified previously in genomes of the human, the mouse, and the rat, and individual kallikrein-like genes have been found in many more species. This study presents the in silico identification of kallikrein gene families in the recently sequenced genomes of four additional mammalian species, the chimpanzee, the dog, the pig, and the opossum. Phylogenies were constructed with gene sequences from all seven mammalian families, using Bayesian analysis, which clarified the evolutionary relationships between these genes. Individual gene sequences, as well as concatenated constructs of multiple sequences, were used. Fifteen kallikrein genes were located in the chimpanzee (Pan troglodytes) genome, while only 14 were identified in the canine (Canis familiaris) genome as no orthologue to human KLK3 was found. Thirteen genes were identified from the pig (Sus scrofa) genome, which lacked homologues to KLK2 and KLK3, and 11 genes, orthologous to human KLK5 through KLK15, were found in the opossum (Monodelphis domestica) genome. No kallikrein genes were identified from the available genome sequences of the chicken (Gallus gallus) or African clawed frog (Xenopus tropicalis). Within the family of kallikreins several subfamilies were suggested by phylogenetic analysis. One consisted of KLK4, KLK5, and KLK14; another of KLK9, KLK11, and KLK15; a third of KLK10 and KLK12; a fourth of KLK6 and KLK13; and finally one of KLK8 and the classical kallikreins (KLK1, KLK2, and KLK3).

Purification and characterization of human kallikrein 11, a candidate prostate and ovarian cancer biomarker, from seminal plasma.

PURPOSE: Preliminary data suggest that hK11 is a novel serum biomarker for prostate and ovarian cancer. To examine the enzymatic characteristics of hK11, we purified and functionally characterized native hK11 from seminal plasma. EXPERIMENTAL DESIGN: hK11 was purified from seminal plasma by immunoaffinity chromatography and characterized by kinetic analysis, electrophoresis, Western blots, and mass spectrometry. RESULTS: hK11 is present in seminal plasma at concentrations ranging from 2 to 37 microg/mL. Using immunoaffinity chromatography and reverse-phase high-performance liquid chromatography, we purified hK11 to homogeneity. In seminal plasma, hK11 is present as a free enzyme of approximately 40 kDa. About 40% of hK11 is enzymatically active, whereas the rest is inactivated by internal cleavage after Arg156 (Genbank accession no. AF164623), which generates two peptides of approximately 20 kDa, connected by internal disulfide bonds. Purified hK11 possesses trypsin-like activity and cleaves synthetic peptides after arginine but not lysine residues. It does not cleave chymotrypsin substrates. Antithrombin, alpha1-antichymotrypsin, alpha2-antiplasmin, and alpha1-antitrypsin have no effect on hK11 activity and do not form complexes with hK11 in vitro. The strongest inhibitor, APMSF, completely inhibited hK11 activity at a concentration of 2.5 mmol/L. Aprotinin and an hK11-specific monoclonal antibody inhibited hK11 activity up to 40%. Plasmin is a strong candidate for cleaving hK11 at Arg156. CONCLUSION: This is the first report on purification and characterization of native hK11. We speculate that hK11, along with other kallikreins, proteases, and inhibitors, participates in a cascade enzymatic pathway responsible for semen liquefaction after ejaculation.

Quantification of human tissue kallikreins in the stratum corneum: dependence on age and gender.

Human tissue kallikreins are a family of 15 trypsin or chymotrypsin-like secreted serine proteases (hK1-hK15). hK5, hK6, hK7, hK8, and hK13 have been identified in the stratum corneum (SC), stratum granulosum, and skin appendages. It has been reported that hK5 and hK7 degrade desmosomes/corneodesmosomes, suggesting that kallikreins are responsible for desquamation. We report the quantification of hK5, hK6, hK7, hK8, hK10, hK11, hK13, and hK14 in the SC by ELISA and their variation among age groups. The total SC trypsin and chymotrypsin-like activities were also measured. The amount of hK7, hK8, and hK11 (ng per mg dry weight) were high, and varied from 6 to 14, hK5 (2.0-4.0) was present at intermediate levels, and hK10 (0.65-1.0), hK14 (0.1-0.3), hK6 (0.1-0.3), and hK13 (0.02-0.1) were present at lower levels. hK6 and hK14 were significantly lower in females between 20 and 59 y. hK5, hK7, hK10, hK11, and hK14 were not significantly different across the age groups. hK8 was lowest at extremes of age (highest at 30-39 y), hK6 was lower at >30 y, and hK13 was lower at >20 y. Overall trypsin-like activity did not differ across age groups but was higher in subjects <11 y. Overall chymotrypsin-like activity was not related to age. In conclusion, we found multiple kallikreins in the SC and suggest that these enzymes may be responsible for desquamation through an enzymatic cascade pathway.

A survey of alternative transcripts of human tissue kallikrein genes.

Alternative splicing is prevalent within the human tissue kallikrein gene locus. Aside from being the most important source of protein diversity in eukaryotes, this process plays a significant role in development, physiology and disease. A better understanding of alternative splicing could lead to the use of gene variants as drug targets, therapeutic agents or diagnostic markers. With the rapidly rising number of alternative kallikrein transcripts, classifying new transcripts and piecing together the significance of existing data are becoming increasingly challenging. In this review, we present a systematic analysis of all currently known kallikrein alternative transcripts. By defining a reference form for each of the 15 kallikrein genes (KLK1 to KLK15), we were able to classify alternative splicing patterns. We identified 82 different kallikrein gene transcript forms, including reference forms. Alternative splicing may lead to the synthesis of 56 different protein forms for KLK1-15. In the kallikrein locus, the majority of alternative splicing events occur within the protein-coding region, and to a lesser extent in the 5' untranslated regions (UTRs). The most common alternative splicing event is exon skipping (35%) and the least common events are cryptic exons (3%) and internal exon deletion (3%). Seventy-six percent of kallikrein splice variants that are predicted to encode truncated proteins are the result of frameshifts. Eighty-nine percent of putative proteins encoded by splice variants are predicted to be secreted. Although several reports describe the identification of kallikrein splice variants and their potential clinical utility, this is the first extensive review on this subject. Accumulating evidence suggests that alternative kallikrein forms could be involved in many pathologic conditions or could have practical applications as biomarkers. The organization and analysis of the kallikrein transcripts will facilitate future work in this area and may lead to novel clinical and diagnostic applications.

Sequence and evolutionary analysis of the human trypsin subfamily of serine peptidases.

Serine peptidases (SP) are peptidases with a uniquely activated serine residue in the substrate-binding site. SP can be classified into clans with distinct evolutionary histories and each clan further subdivided into families. We analyzed 79 proteins representing the S1A subfamily of human SP, obtained from different databases. Multiple alignment identified 87 highly conserved amino acid residues. In most cases of substitution, a residue of similar character was inserted, implying that the overall character of the local region was conserved. We also identified several conserved protein motifs. 7-13 cysteine positions, potentially forming disulfide bridges, were also found to be conserved. Most members are secreted as inactive (pro) forms with a trypsin-like cleavage site for activation. Substrate specificity was predicted to be trypsin-like for most members, with few chymotrypsin-like proteins. Phylogenetic analysis enabled us to classify members of the S1A subfamily into structurally related groups; this might also help to functionally sort members of this subfamily and give an idea about their possible functions.

Genomic overview of serine proteases.

Serine proteases (SP) are peptidases with a uniquely activated serine residue in the substrate-binding pocket. They represent about 0.6% of all proteins in the human genome. SP are involved in many vital functions such as digestion, blood clotting, fibrinolysis, fertilization, and complement activation and are related to many diseases including cancer, arthritis, and emphysema. In this study, we performed a genomic analysis of human serine proteases utilizing different databases, primarily that of MEROPS. SP are distributed along all human chromosomes except 18 and Y with the highest density (23 genes) on chromosome 19. They are either randomly located within the genome or occur in clusters. We identified a number of SP clusters, the largest being the kallikrein cluster on chromosome 19q13.4 which is formed of 15 adjacent genes. Other clusters are located on chromosomes 19p13, 16p13, 14q11, 13q35, 11q22, and 7q35. Genes of each cluster tend to be of comparable sizes and to be transcribed in the same direction. The members of some clusters are sometimes functionally related, e.g., the involvement of many kallikreins in endocrine-related malignancies and the hematopoietic cluster on chromosome 14. It is hypothesized that members of some clusters are under common regulatory mechanisms and might be involved in cascade enzymatic pathways. Several functional domains are found in SP, which reflect their functional diversity. Membrane-type SP tend to cluster in 3 chromosomes and have some common structural domains. Several databases are available for screening, structural and functional analysis of serine proteases. With the near completion of the Human Genome Project, research will be more focused on the interactions between SP and their involvement in pathophysiological processes.