Åke Lundwall

Organization and evolution of the glandular kallikrein locus in Mus musculus

The gene of tissue kallikrein and closely related genes constitute the glandular kallikrein (GK) gene family. The number of members varies between species, ranging from three human to 25 murine. Recently, the gene family was extended with 12 new members, KLK4-KLK15, that were identified adjacent to the classical GK genes on human chromosome 19. In this report, the structure and phylogeny of the mouse GK gene locus are described. A comparison of the human and murine loci shows that the locations of the tissue kallikrein gene and KLK4-KLK15 are conserved. The region between the tissue kallikrein gene and KLK15, devoid of genes in human, is expanded and contains 23 classical GK genes in mouse. Downstream of KLK15, where the genes encoding PSA and hK2 are located in human, mouse carries the pseudogene PsimGK25. Phylogenetic analyses show that classical GK genes emerged after the separation of the primate and rodent lineages, forming a subgroup within the newly extended GK family.

Molecular cloning of a small prostate protein, known as beta-microsemenoprotein, PSP94 or beta-inhibin, and demonstration of transcripts in non-genital tissues

In order to study the gene expression of the seminal plasma protein beta-microseminoprotein, also known as PSP94 and beta-inhibin, clones encoding this protein were isolated from a cDNA library constructed in lambda gt11. Nucleotide sequencing confirmed the structure of a previously cloned cDNA. By northern blot analysis identical sized transcripts were demonstrated in the prostate, the respiratory (tracheal, bronchial and lung) tissues and the antrum part of the gastric mucosa. Thus, the protein is not primarily associated with male reproductive function. Although probably of no physiological significance, a slight structural similarity to the ovarian inhibin beta-chains was identified in the C-terminal half of the molecule.

Expression of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) in ileum and other extraprostatic tissues

Prostate‐specific antigen (PSA) is a widely used marker for prostate cancer. In the literature, there are reports of nonprostatic expression of PSA that potentially can affect early diagnosis. However, the results are scattered and inconclusive, which motivated us to conduct a more comprehensive study of the tissue distribution of PSA and the closely related protein human glandular kallikrein 2 (hK2). RT‐PCR, in situ hybridization and immunohistochemistry were used to detect expression of both PSA and hK2 in secretory epithelial cells of trachea, thyroid gland, mammary gland, salivary gland, jejunum, ileum, epididymis, seminal vesicle and urethra, as well as in Leydig cells, pancreatic exocrine glands and epidermis. Immunometric measurements revealed that the concentration of PSA in nonprostatic tissues represents less than 1% of the amount in normal prostate. Pronounced expression of PSA was detected in the Paneth cells in ileum, which prompted us to compare functional parameters of PSA in ileum and prostate. We found that in homogenates from these 2 tissues, PSA manifested equivalent amidolytic activity and capacity to form complexes with protease inhibitors in blood in vitro. Thus, PSA released from sources other than the prostate may add to the plasma pool of this protein, but given the lower levels detected from those sites, it is unlikely that nonprostatic PSA normally can interfere with the diagnosis of prostate cancer. Nevertheless, this risk should not be neglected as it may be of clinical significance under certain circumstances. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html.

Evolution of kallikrein-related peptidases in mammals and identification of a genetic locus encoding potential regulatory inhibitors

Abstract The human kallikrein locus on chromosome 19 consists of 15 genes encoding serine proteases. Here we review studies on their evolution, which demonstrate that there are several taxon-unique KLK1 paralogs in mouse, rat and horse, but not in primates and many other mammals. Furthermore, the duplication yielding KLK2 and prostate-specific antigen (KLK3) appears to be specific to primates, but a functional progenitor to KLK2 is expressed in the dog prostate. The linkage to a locus of possible regulatory protease inhibitors on chromosome 20 is discussed, as is the potential role of the kallikrein locus in innate immunity.

A highly conserved protein secreted by the prostate cancer cell line PC-3 is expressed in benign and malignant prostate tissue

Abstract In this study we characterize a novel gene on human chromosome 9 and its translation product, PC3-secreted microprotein (PSMP). The gene contains three exons that encode a protein of 139 amino acid residues, including a predicted signal peptide of 36 residues. The molecule is homologous to β-microseminoprotein (MSP), a protein of unknown function, secreted at high concentration by the prostate gland. These two proteins have only 23% sequence identity, but their common origin is revealed by a preserved pattern of Cys residues. In contrast to MSP, which shows poor conservation between species, PSMP is very conserved. High transcript levels were detected in the prostate cancer cell line PC-3. Antiserum raised against PSMP detected a protein with an apparent molecular mass of 18 kDa in culture medium conditioned by PC-3 cells, but in cell lysates the antiserum also recognized a molecular species of 16 kDa, suggesting that PSMP undergoes post-translational modification. Xenografted PC-3 cell tumors in athymic nude mice showed strong staining for both PSMP protein and mRNA. Studies on human prostate cancer specimens showed immunohistochemical staining of both tumor and benign glandular cells. Our results suggest that PSMP is an important protein with significance in prostate cancer.

Glandular kallikreins of the cotton-top tamarin: molecular cloning of the gene encoding the tissue kallikrein

The glandular kallikrein family is composed of structurally related serine proteases. Studies show that the mouse family encompasses at least 14 highly conserved functional genes, but of these only the tissue kallikarein has a human ortholog. In man, the tissue kallikrein display high sequence similarity with prostate specific antigen and human glandular kallikrein 2, suggesting that they evolved after the separation of primates and rodents. A phylogenetic study of the genes encoding glandular kallikreins in species evolutionarily located between rodents and man may reveal interesting details on how the gene family evolved, which in turn could yield information about the function of the proteins. Therefore, we have initiated a study of the glandular kallikreins of the cotton-top tamarin (Saguinus oedipus), a New World Monkey. Here, we report the cloning and nucleotide sequence of one of these, the tissue kallikrein gene. The gene of 4.4 kb is composed of five exons, and the structure is 90% similar to that of the orthologous human gene. It gives rise to a polypeptide of 261 amino acids, including a signal peptide of 17 residues, a pro-piece of 7 residues, and the mature protein of 237 residues with an estimated molecular mass of 26.3 kD. The similarity to the human prostate specific antigen and human glandular kallikrein 2 genes is 73% and 72%, respectively, including introns and flanking regions. The lower similarity to these genes compared with the human tissue kallikrein gene indicates that they, or a progenitor to them, arose in primates prior to the separation of New and Old World monkeys. Genomic Southern blots also show that the cotton-top tamarin genome encompasses at least one more glandular kallikrein gene.

Rapidly evolving marmoset MSMB genes are differently expressed in the male genital tract

BackgroundBeta-microseminoprotein, an abundant component in prostatic fluid, is encoded by the potential tumor suppressor gene MSMB. Some New World monkeys carry several copies of this gene, in contrast to most mammals, including humans, which have one only. Here we have investigated the background for the species difference by analyzing the chromosomal organization and expression of MSMB in the common marmoset (Callithrix jacchus).MethodsGenes were identified in the Callithrix jacchus genome database using bioinformatics and transcripts were analyzed by RT-PCR and quantified by real time PCR in the presence of SYBR green.ResultsThe common marmoset has five MSMB: one processed pseudogene and four functional genes. The latter encompass homologous genomic regions of 32-35 kb, containing the genes of 12-14 kb and conserved upstream and downstream regions of 14-19 kb and 3-4 kb. One gene, MSMB1, occupies the same position on the chromosome as the single human gene. On the same chromosome, but several Mb away, is another MSMB locus situated with MSMB2, MSMB3 and MSMB4 arranged in tandem. Measurements of transcripts demonstrated that all functional genes are expressed in the male genital tract, generating very high transcript levels in the prostate. The transcript levels in seminal vesicles and testis are two and four orders of magnitude lower. A single gene, MSMB3, accounts for more than 90% of MSMB transcripts in both the prostate and the seminal vesicles, whereas in the testis around half of the transcripts originate from MSMB2. These genes display rapid evolution with a skewed distribution of mutated nucleotides; in MSMB2 they affect nucleotides encoding the N-terminal Greek key domain, whereas in MSMB3 it is the C-terminal MSMB-unique domain that is affected.ConclusionCallitrichide monkeys have four functional MSMB that are all expressed in the male genital tract, but the product from one gene, MSMB3, will predominate in seminal plasma. This gene and MSMB2, the predominating testicular gene, have accumulated mutations that affect different parts of the translation products, suggesting an ongoing molecular specialization that presumably yields functional differences in accessory sex glands and testis.