Evolutionary history of tissue kallikreins.

The gene family of human kallikrein-related peptidases (KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs.

[Molecular forms of PSA]. / Les formes moléculaires du PSA.

Molecular forms of serum PSA (prostate specific antigen) have been developped to improve total PSA sensitivity and specificity in prostate cancer diagnosis and staging. Total PSA is measured in bound (complexed PSA) and unbound (free PSA) molecular forms. Their levels in absolute values and in relation to total PSA (f/t PSA and c/t PSA) have been evaluated. The percentage of free PSA is more specific but less sensitive than tPSA and it is not recommended as a first line diagnostic test. It may be useful as a second-line test, prescribed by the urologist after a first series of negative biopsies. There is general agreement that at high sensitivity, cPSA provides higher specificity compared with tPSA in the gray zone (2-10 ng/ml). Nevertheless the widespread use of tPSA an the small benefit in terms of specificity explains why cPSA is not generally recommanded. Molecular derivates of free PSA have been identified: proPSA (precursor inactive form of PSA), intact PSA (an additionnal form of proPSA that is found intact and inactive), human Kallikrein 2 and BPSA (for benign PSA wich is associated to BPH) have been evaluated. Preliminary studies did not have demonstrate their ability to discriminate between cancer and BPH, and did not define cutoff values.

Proteolytic processing of human growth hormone by multiple tissue kallikreins and regulation by the serine protease inhibitor Kazal-Type5 (SPINK5) protein.

BACKGROUND: Human growth hormone (hGH) is naturally present in numerous isoforms, some of which arise from proteolytic processing in both the pituitary and periphery. The nature of the enzymes that proteolytically cleave hGH and the regulation of this process are not fully understood. Our objective is to examine if members of a newly discovered human tissue kallikrein family (KLKs) are expressed in the pituitary and if these enzymes can cleave hGH in-vitro. METHODS: Expression of 12 of the KLKs (KLKs 4-15) and serine protease inhibitor Kazal-type 5 (SPINK5) genes and their proteins in the pituitary was examined by RT-PCR and immunohistochemistry. Recombinant hGH was digested by various recombinant KLKs and fragments were characterized by N-terminal sequencing. SPINK5 recombinant fragments were used for inhibition of KLK activities. RESULTS: We here describe for the first time expression of numerous KLKs (KLKs 5-8, 10-14) and SPINK5 in the pituitary. KLK6 and SPINK5 appeared to be localized to hGH-producing cells. KLKs 4-6, 8, 13 and 14 were able to cleave hGH, yielding various isoforms, in vitro. Inhibitor SPINK5 fragments were able to suppress activity of KLKs 4, 5 and 14 in vitro. Based on these data, we propose a model for the proteolytic processing of hGH in the pituitary and the regulation of this system by SPINK5 inhibitory domains. We speculate that loss of SPINK5 inhibitory domains, as in the case of Netherton syndrome, may lead to proteolytic over-processing of hGH and to growth retardation. CONCLUSION: We conclude that many KLKs and SPINK5 are expressed in the pituitary. This serine protease-inhibitor system is likely to participate in the regulated proteolytic processing of hGH in the pituitary, leading to generation of hGH fragments. Our data suggest that KLKs 5, 6 and 14 might be involved in this process.

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.

The tissue kallikrein family of serine proteases: functional roles in human disease and potential as clinical biomarkers.

Prostate specific antigen (PSA) or human kallikrein 3 (hK3) has long been an effective biomarker for prostate cancer. Now, other members of the tissue kallikrein (KLK) gene family are fast becoming of clinical interest due to their potential as prognostic biomarkers. particularly for hormone dependent cancers. The tissue kallikreins are serine proteases that are encoded by highly conserved multi-gene family clusters in rodents and humans. The rat and mouse loci contain 10 and 25 functional genes, respectively, while the human locus at 19q 13.4 contains 15 genes. The structural organization and size of these genes are similar across species; all genes have 5 coding exons that encode a prepro-enzyme. Although the physiological activators of these zymogens have not been described, in vitro biochemical studies show that some kallikreins can auto-activate and others can activate each other, suggesting that the kallikreins may participate in an enzymatic cascade similar to that of the coagulation cascade. These genes are expressed, to varying degrees, in a wide range of tissues suggesting a functional involvement in a diverse range of physiological and pathophysiological processes. These include roles in normal skin desquamation and psoriatic lesions, tooth development, neural plasticity, and Alzheimer's disease (AD). Of particular interest is the expression of many kallikreins in prostate, ovarian, and breast cancers where they are emerging as useful prognostic indicators of disease progression.

[Serum markers for early detection and staging of prostate cancer. Status report on current and future markers]. / Serummarker in der Früherkennung und dem Staging des Prostatakarzinoms. Eine Bestandsaufnahme aktueller und zukünftiger Marker.

Prostate-specific antigen (PSA) is by far the most important tumor marker in urology and has revolutionized early detection, staging, treatment, and aftercare of prostate cancer [77]. Despite these merits, inadequacies have surfaced which prohibit characterizing PSA as a perfect tumor marker. First, PSA is not a marker for prostate cancer as such:benign prostate hyperplasia, prostatitis [40,69], or prostatic manipulation [66] influence serum concentrations of PSA and lead to biopsies that are costly and potentially harmful. In the entire PSA range between 4 and 10 ng/ml, the specificity at a sensitivity of 95% continues to remain unsatisfactory. Furthermore, 30-40% of all men develop prostate cancer, but only 9-11% a clinically significant tumor burden, and 2.5-4.3% of all men die from prostate cancer. The vast majority of all carcinomas are thus in significant in terms of the patient's life expectancy. PSA is incapable of differentiating these clinically insignificant carcinomas from significant ones. Finally, prevalence of prostate cancer is increasing due to higher life expectancy. On the other hand, particularly patients aged 50-70 years are the ones who develop an aggressive form of carcinoma and profit from early detection and treatment. The global term "total PSA"encompasses a heterogeneous blend of bound and free molecular forms of PSA. Complexed PSA represents the major form of total PSA. The smaller portion, free PSA, is enzymatically inactive. In addition, different isoforms of free PSA exist Recent studies provide support for clinical application of these isoforms for early detection of prostate cancer. Clinical measurement of human glandular kallikrein 2 (hK2) serves as a complementary marker to PSA for early detection of prostate cancer and constitutes a considerable improvement over PSA as a staging marker for clinically localized prostate cancer. This overview summarizes established and potentially new forms of PSA and hK2 for early detection and staging of prostate cancer.

The new human tissue kallikrein gene family: structure, function, and association to disease.

The human tissue kallikrein gene family was, until recently, thought to consist of only three genes. Two of these human kallikreins, prostate-specific antigen and human glandular kallikrein 2, are currently used as valuable biomarkers of prostatic carcinoma. More recently, new kallikrein-like genes have been discovered. It is now clear that the human tissue kallikrein gene family contains at least 15 genes. All genes share important similarities, including mapping at the same chromosomal locus (19q13.4), significant homology at both the nucleotide and protein level, and similar genomic organization. All genes encode for putative serine proteases and most of them are regulated by steroid hormones. Recent data suggest that at least a few of these kallikrein genes are connected to malignancy. In this review, we summarize the recently accumulated knowledge on the human tissue kallikrein gene family, including gene and protein structure, predicted enzymatic activities, tissue expression, hormonal regulation, and alternative splicing. We further describe the reported associations of the human kallikreins with various human diseases and identify future avenues for research.