Human kallikrein 4: enzymatic activity, inhibition, and degradation of extracellular matrix proteins.

Human kallikrein 4 (hK4) is a member of the expanded family of human kallikreins, a group of 15 secreted proteases. While this protein has been associated with ovarian and prostate cancer prognosis, only limited functional information exists. Therefore, we have undertaken an investigation of its enzymatic properties regarding substrate preference, degradation of extracellular matrix proteins, and its inhibition by various inhibitors. We successfully expressed and purified active recombinant hK4 from supernatants of the Pichia pastoris expression system. This enzyme seems to cleave more efficiently after Arg compared to Lys at the P1 position and exhibits modest specificity for amino acids at positions P2 and P3. hK4 forms complexes with alpha1-antitrypsin, alpha2-antiplasmin and alpha2-macroglobulin. The protease mediates limited degradation of extracellular matrix proteins such as collagen I and IV, and more efficient degradation of the alpha-chain of fibrinogen. The cleavage of extracellular matrix proteins by hK4 suggests that this enzyme may play a role in tissue remodeling and cancer metastasis.

Prognostic implications of the immunohistochemical expression of human kallikreins 5, 6, 10 and 11 in renal cell carcinoma.

Human kallikreins 5, 6, 10 and 11 (hK5, 6, 10 and 11) are expressed by many normal tissues, and it has been suggested that they may represent candidate tumor-diagnostic or -prognostic markers. In patients with renal cell carcinoma (RCC), outcome is unpredictable despite the use of conventional prognostic factors. The aim of this study is to evaluate the immunohistochemical expression and the prognostic value of the above kallikreins in RCC. The study comprised 95 patients who underwent radical nephrectomy for RCC. The median follow-up period was 60 months (range 1-180 months). Fifty-seven RCC cases were immunostained for hK5, 70 for hK6, 70 for hK10 and 69 for hK11. The streptavidin-biotin-peroxidase method of immunostaining was performed using anti-hK5, anti-hK6, anti-hK10 and anti-hK11 monoclonal and polyclonal antibodies. The immunohistochemical expression of these kallikreins was correlated with tumor size, histological type, histological malignancy according to the Fuhrman four-grade scale, mitotic index, pathological stage and disease survival. For the statistical analysis, four grades were collapsed into two by which RCC cases were categorized as low malignant (LM) and high malignant (HM). In the normal renal parenchyma adjacent to the tumors, the renal tubular epithelium showed a cytoplasmic expression of all four kallikreins. In RCC, immunohistochemical expression was decreased: 33 of 57 cases (58%) were positive for hK5, 27 of 70 (39%) for hK6, 46 of 70 (66%) for hK10 and 32 of 69 (46%) for hK11. A statistically significant positive correlation was observed among the immunohistochemical expression of all kallikreins. HM-RCC expressed all kallikreins in a higher percentage of cases than LM-RCC, but statistically significant differences were only observed for hK6 and hK10 (55 vs. 27%, p = 0.016, and 79 vs. 56%, p = 0.044, respectively). hK6 and hK11 expression showed a positive correlation to pathological stage: hK6 with both Robson and TNM 2002 staging systems (p = 0.010 and p = 0.017, respectively), and hK11 only with the Robson staging system (p = 0.045). In both the Kaplan-Meier and the univariate Cox regression analyses, hK6 expression was negatively correlated with disease-specific survival (p = 0.05 and p = 0.038, respectively). In univariate analysis, nuclear grade, Robson stage and TNM stage also correlated with disease-specific survival. However, in the multivariate analysis, TNM stage was the only independent prognostic factor. In conclusion, although the immunohistochemical expression of hK5, hK6, hK10 and hK11 was downregulated in RCC, tumors of high grade and late stage expressed one or more of the above kallikreins in a higher percentage of cases, and hK6 may predict a poor disease outcome in RCC.

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.

Human kallikrein 4: quantitative study in tissues and evidence for its secretion into biological fluids.

BACKGROUND: Human kallikrein 4 (hK4) is a proteolytic enzyme belonging to the tissue kallikrein family of serine proteases. Previous tissue expression studies have demonstrated highest KLK4 mRNA expression in prostatic tissue, but there has been only limited evidence for the presence of hK4 protein in prostate and other tissues and in corresponding biological secretions. METHODS: To investigate the concentrations of hK4 in tissues and biological fluids, we developed a new hK4-specific sandwich-type immunoassay using a monoclonal antibody as the capture reagent. RESULTS: The assay has a detection limit of 0.02 microg/L and <0.1% cross-reactivity toward any of the other 14 human kallikreins. Twelve of 40 tissue extracts prepared from various human tissues contained detectable hK4 concentrations (0.68-7143 ng/g of total protein), with healthy prostate tissue containing the highest amount of hK4. Examination of 16 malignant and 18 benign prostate tissues revealed no significant differences in hK4 protein content, and the tissues contained a wide range of values (benign, <0.02 to 801 ng/g; malignant, <0.02 to 824 ng/g). Among the biological fluids tested, seminal plasma and urine contained widely varying amounts of hK4; concentrations in 54 urine samples were <0.02 to 2.6 microg/L, whereas concentrations in 58 seminal plasma samples were 0.2-202 microg/L. Affinity purification of hK4 from seminal plasma and subsequent mass spectrometry demonstrated the secreted nature of hK4 in seminal plasma. CONCLUSIONS: hK4 is found primarily in prostate tissue and is secreted in seminal plasma. Its value as a novel prostatic biomarker needs to be defined further.

Cloning, physical mapping and structural characterization of the human alpha(A)-adaptin gene.

Adaptins are major structural components of heterotetrameric protein complexes called adaptors, which are involved in intracellular receptor transport via clathrin-coated vesicles. In mice, one of these adaptins has been shown to be encoded by two genes, alpha(A)-adaptin and alpha(C)-adaptin, the former of which is expressed as two alternatively spliced transcripts. Using positional cloning gene approaches, we were able to identify the human alpha(A)-adaptin gene, which consists of 24 exons spanning over 40 kb on chromosome 19q13.3 between the loci of the R-ras gene and the polynucleotide kinase phosphatase gene. The novel gene encodes a 977 amino acid, 107.6 kDa protein with 98% amino acid sequence identity to its murine ortholog. Human alpha(A)-adaptin is expressed as a full-length transcript in forebrain, skeletal muscle, spinal cord, cerebellum, salivary gland, heart and colon. It is also ubiquitously expressed in tissues and in ZR-75-1 breast cancer cells and LNCaP prostate carcinoma cells as a smaller variant generated by splicing out of an exon encoding 22 amino acids in the hinge region of the protein.