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.

New world, but not Old World, monkeys carry several genes encoding beta-microseminoprotein.

It was shown by Southern hybridization that cotton-top tamarin and common marmoset, New World monkeys, carry three or more genes encoding beta-microseminoprotein, also known as PSP94. In contrast, the genomes of Old World monkeys, as represented by rhesus macaque and sacred baboon, contain a single gene. Clones containing three different genes encoding beta-microseminoprotein were isolated from a cotton-top tamarin genomic library. They carry two complete genes of four exons and a third gene lacking the first exon. The structure suggests that the three genes are functionally active and give rise to transcripts that are approximately 86% similar in sequence. By sequencing one gene in full, it was shown that the introns carry an excess of interspersed repeats, on average 29% of the introns consist of Alu repeats. A phylogenetic analysis demonstrated that the genes probably arose in New World monkeys after the separation from Old World primates.

Measurement of prostate-specific antigen and human glandular kallikrein 2 in different body fluids.

It has been demonstrated that prostate-specific antigen (PSA), in spite of its name, can be detected in body fluids and tumors from a variety of organs. Investigations have shown that human glandular kallikrein 2 (hK2), a related prostate-secreted protease, can activate the zymogen form of PSA, suggesting that the two enzymes might work as a functional unit, with hK2 as the activator molecule and PSA as the effector molecule. If this is true, then hK2 should be found together with PSA in body fluids other than seminal plasma, as well. Recently, a sensitive and specific assay was devised for hK2, enabling its measurement in picogram quantities. With this assay, the concentration of hK2 was determined in samples of seminal plasma, amniotic fluid, breast milk, and saliva. Simultaneously, the samples were assayed for molecular forms of PSA. In seminal plasma, the mean PSA concentration was 0.82 mg/ml, while the hK2 level was around two orders of magnitude lower: mean value, 6.4 microg/ml. Approximately the same ratio of PSA to hK2 as in seminal plasma was found in amniotic fluid and breast milk, but in most samples, the hK2 values were too low for direct measurements and had to be concentrated prior to analysis. Measurable levels of PSA, all in the free form, were detected in amniotic fluid at the thirteenth week of gestation and then gradually increased to levels around and over 1 microg/L from the twentieth week. Significant levels of PSA were detected in amniotic fluid collected at delivery, also. Measurable levels of mammary PSA were primarily detected in colostrum, with a range from less than 0.03 microg/L to 2.1 mg/L. Around half of the molecules were in complex with protease inhibitor. Most surprisingly, determinations on saliva samples showed that none of them had detectable PSA levels but had measurable concentrations of hK2 with a mean value, 0.09 microg/L. The presence in saliva suggests that hK2 can be the human equivalent to one of the mouse salivary kallikreins with important biological function, like the epidermal growth factor-binding protein or the gamma subunit of nerve growth factor. However, this was ruled out, as a phylogenetic analysis showed that the human and mouse glandular kallikreins evolved independently from a common precursor after the separation of the primate and rodent lineages. In conclusion, the measurements show that in addition to the previously known secretion in seminal plasma, hK2 is secreted in amniotic fluid, breast milk, and saliva. Furthermore, the concerted expression of PSA and hK2 in seminal plasma, amniotic fluid, and breast milk suggests that the two proteases might form a functional unit but not always as demonstrated by the sole presence of hK2 in saliva.