Structure of mouse kallikrein gene family suggests a role in specific processing of biologically active peptides

Mason, Anthony J.; Evans, Bronwyn A; Cox, David; Shine, John; Richards, Robert I.

doi:10.1038/303300a0

Cited by 295 publications

(138 citation statements)

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“…8). Phylogenetic analysis separated the classical kallikreins (KLK1, KLK2, and PSA) and grouped KLK-L4 with zyme, TLSP, KLK-L3, neuropsin, and NES1 genes, consistent with previously published studies (8,24,25) and indicating that this group of genes probably arose from a common ancestral gene by duplication.…”

Section: Genesupporting

confidence: 72%

Identification and Characterization of KLK-L4, a New Kallikrein-like Gene That Appears to be Down-regulated in Breast Cancer Tissues

Yousef¹,

Chang²,

Diamandis³

2000

Journal of Biological Chemistry

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Kallikreins are a subgroup of serine proteases and these proteolytic enzymes have diverse physiological functions in many tissues. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins constitute a large multigene family, but in humans, only three genes were identified. By using the positional candidate gene approach, we were able to identify a new kallikrein-like gene, tentatively named KLK-L4 (for kallikrein-like gene 4). This new gene maps to chromosome 19q13.3-q13.4, is formed of five coding exons and four introns, and shows structural similarity to other kallikreins and kallikrein-like genes. KLK-L4 is expressed in a variety of tissues including prostate, salivary gland, breast, and testis. Our preliminary results show that KLK-L4 is down-regulated, at the mRNA level, in breast cancer tissues and breast cancer cell lines. Its expression is regulated by steroid hormones in the breast cancer cell line BT-474. This gene may be involved in the pathogenesis and/or progression of breast cancer and may find applicability as a novel cancer biomarker.Prostate-specific antigen (PSA) 1 testing has revolutionized the management of patients with prostate cancer (1). The PSA gene (KLK3) is a member of the human tissue kallikrein gene family, which is also comprised of human glandular kallikrein 2 (KLK2) (2) and pancreatic/renal kallikrein (KLK1) (3) genes. More recently, new serine proteases with a high degree of homology to the kallikrein genes were cloned (4 -9). The successful diagnostic use of PSA in prostate cancer suggests that other related or unrelated molecules might be discovered and serve as diagnostic tests for breast, ovarian, and other cancers. In addition to PSA, human glandular kallikrein 2 (encoded by the KLK2 gene) may be useful as an adjuvant diagnostic marker for prostate cancer (10). Accumulating evidence indicates that some members of the kallikrein gene family are implicated in carcinogenesis. The normal epithelial cell-specific 1 gene (NES1) was found to be a tumor suppressor (11) that is down-regulated during breast cancer progression. The zyme/ protease M/neurosin gene is expressed in primary breast cancers but is down-regulated at metastatic sites (4).The large size of the kallikrein gene family in other species, such as rat and mouse, where kallikreins are encoded by 13-24 genes (12-13), and the recent identification of new kallikreinlike genes suggested that the human kallikrein gene family may be larger than previously thought. The rodent kallikrein genes are located in clusters on chromosome 7, and the region between two mouse kallikrein genes in a cluster can be as small as 3-7 kb (14).In our efforts to identify new kallikrein-like genes that might be useful as diagnostic and/or prognostic markers for cancer, we studied a genomic area of ϳ300 kb around chromosome 19q13.3-q13.4, where the known human kallikrein genes are localized. We were able to identify three new kallikrein-like genes; KLK-L1 (for kallikrein-like gene 1) (15) KLK-L2 (fo...

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Section: Genesupporting

confidence: 72%

Identification and Characterization of KLK-L4, a New Kallikrein-like Gene That Appears to be Down-regulated in Breast Cancer Tissues

Yousef¹,

Chang²,

Diamandis³

2000

Journal of Biological Chemistry

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“…Heavily boxed areas contain residues neighboring the vicinity of the active sites of all serine proteases and are highly homologous. Areas that are lightly boxed indicate regions believed to line the substrate binding pocket (20). Residues underlined, numbering 45 and 69-71 of PA, represent potential attachment sites for carbohydrate.…”

Section: Methodsmentioning

confidence: 99%

Human prostate-specific antigen: structural and functional similarity with serine proteases.

Watt¹,

Lee²,

M'Timkulu³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

497

255

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The complete amino acid sequence of the prostate-specific antigen (PA) from human seminal plasma has been determined from analyses of the peptides generated by cyanogen bromide, hydroxylamine, endoproteinases Arg-C and Lys-C. The single polypeptide chain of PA contains 240-amino acid residues and has a calculated Mr of 26,496. An N-linked carbohydrate side chain is predicted at asparagine-45, and O-linked carbohydrate side chains are possibly attached to serine-69, threonine-70, and serine-71. The primary structure of PA shows a high degree of sequence homology with other serine proteases ofthe kallikrein family. The active site residues of histidine, aspartic acid, and serine comprising the chargerelay system of typical serine proteases were found in similar positions in PA (histidine-41, aspartic acid-96, and serine-192). At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant interleukin 2, and-to a lesser extent-gelatin, myoglobin, ovalbumin, and fibrinogen. The cleavage sites of these proteins by PA were chemically analyzed as the a-carboxyl side of some hydrophobic residues, tyrosine, leucine, valine, and phenylalanine, and of basic residues histidine, lysine, and arginine. The chymotrypsin-like activity of PA exhibited with the chromo- (6) and was tested as a marker for postcoital detection in rape investigations (7). Although the clinical utility of PA has been shown, its biological function and chemical structure are not well characterized (8). We report here the complete amino acid sequence of PA and describe the characteristics of its enzymatic properties. MATERIALS AND METHODSPA was purified from human seminal plasma as described (1). PA was finally purified on a large-pore Vydac (Hesperia, CA) C4 column and eluted either with a linear gradient between 70% (vol/vol) buffer A (buffer A = 0.1% trifluoroacetic acid in H20) and 37% (vol/vol) buffer B (buffer B = 0.1% trifluoroacetic acid in acetonitrile) in 280 min or 10% (vol/vol) buffer B and 80% (vol/vol) buffer B in 60 min.The enzymatic activity of PA was assessed on commercially purified proteins including insulin A, insulin B, gelatin, myoglobin, ovalbumin, fibrinogen (Sigma), and recombinant interleukin 2 (Ala-IL2, Cetus). The substrates (1 mg/ml) were dissolved in either 0.1 M ammonium bicarbonate or 50 mM Tris HCl, pH 7.8, containing PA at 0.1 mg/ml. In some cases, a mass ratio (enzyme:substrate) of 1:20 was used. After an 18-hr digestion at 37°C, the peptides were separated by HPLC using a 60-min linear gradient of 0-80% (vol/vol) buffer B. To determine the peptide bond specificity of PA, hydrazinolysis was performed on each ofthe PA/substrate digestion mixtures, substrate alone, and intact PA as described (9). The free carboxyl-terminal amino acids were lyophilized and analyzed on a Beckman 121MB amino acid analyzer.The kinetics ofPA hydrolytic activity on synthetic substrates were studied by monitoring the absorbance change at room temperature using a Hewlett-Packard 8450A spectrophotometer. The following substrates were used: N...

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“…More gene copies per cell means more templates available for synthesis of mRNA transcripts. We investigated this possibility in connection with collagenase because of indications in the literature that phorbol esters might induce gene amplification (56), and our own observation that even confluent rabbit synovial fibroblasts undergo further DNA synthesis after PMA treatment (3). However, after induction of new collagenase synthesis with PMA, the collagenase loci were not found to be amplified.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Rabbit Genes for Synovial Cell Collagenase

Fini

Gross

Brinckerhoff

1986

Arthritis & Rheumatism

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To provide tools for understanding collagenase gene expression in rheumatoid arthritis, we have isolated and characterized genomic clones for rabbit synovial cell collagenase. These clones represent 2 types of collagenase gene, at least 1 of which is transcribed in synovial fibroblasts. By examining the rabbit genome in situ, we provide evidence that there are only 2 different synovial cell collagenase genes found in a haploid genome. Amplification of these genes is not a mechanism far collagenase messenger RNA induction by phorbol esters.The metalloproteinase, collagenase, plays a primary role in the joint destruction seen in rheumatoid arthritis (1). Vast excesses of this enzyme are secreted by the fibroblasts of the rheumatoid pannus. The fact that collagen breakdown can be initiated only by collagenase makes this enzyme rate-limiting in collagenolysis. One approach to the successful control of collagenolysis in diseases such as rheumatoid arthritis may lie in inhibiting the production of collagenase. Thus, an understanding of mechanisms regulating Studies in our laboratory, designed to understand the control of collagenase secretion, have been facilitated by the development of a cell culture system of synovial fibroblasts from rabbits (2). These cultures secrete negligible levels of collagenase when untreated. However, they can be stimulated to secrete major amounts of collagenase by treatment with numerous agents, including the tumor promoter, phorbol myristate acetate (PMA) (3), and crystals of monosodium urate monohydrate (4). Furthermore, additional agents such as retinoic acid or dexamethasone can reverse or suppress induction (5). To date, experiments with human synovial cells have corroborated our findings with the rabbit cell studies, and have thus validated the rabbit system as a model (6,7).Earlier studies demonstrated that inducers act to increase the synthesis of new collagenase protein rather than stimulate the release of previously synthesized enzyme stored intracellularly (8). With the development of a complementary DNA (cDNA) clone for collagenase, we showed that on induction, an increase in immunoreactive collagenase secreted into the culture medium is directly correlated with increased collagenase messenger RNA (mRNA) levels (9). Several steps in RNA metabolism could contribute to the increase in collagenase message levels, including increased transcription from the collagenase gene(s), greater mRNA stability, or changes in processing of pre-mRNA to mRNA. To study the contribution of these processes to mRNA induction, as well as the mechanisms by which they operate, requires more than a cDNA clone, which is merely a copy of mRNA. The collagenase gene itself must be isolated

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Structure of mouse kallikrein gene family suggests a role in specific processing of biologically active peptides

Cited by 295 publications

References 29 publications

Identification and Characterization of KLK-L4, a New Kallikrein-like Gene That Appears to be Down-regulated in Breast Cancer Tissues

Identification and Characterization of KLK-L4, a New Kallikrein-like Gene That Appears to be Down-regulated in Breast Cancer Tissues

Human prostate-specific antigen: structural and functional similarity with serine proteases.

Characterization of Rabbit Genes for Synovial Cell Collagenase

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