Microheterogeneity of rat submaxillary gland kallikrein k10, a member of the kallikrein family

A tissue-kallikrein-related proteinase present in rat submaxillary glands, which was previously called endopeptidase k, has been further characterized and compared with other members of the kallikrein family. The partial primary structure of this proteinase, now called kallikrein k10, is very similar to that of proteinase B [Kato, H., Nakanishi, E., Enjyoji, K., Hayashi, I., Oh-Ishi, S. & Iwanaga, S. (1987) J. Biochem. (Tokyo) 102, 1389-1404] and T-kininogenase [Xiong, W., Chen. L. M. & Chao, J. (1990) J. Biol. Chem. 265, 2822-2827], but no corresponding gene or mRNA has so far been found. Kallikrein k10 is microheterogeneous due to variable glycosylation of its N-terminal light chain and to variable processing at its kallikrein loop, as shown by endo-beta-N-acetylglucosaminidase F treatment, amino acid sequence analysis and mass spectrometry. The enzymatic properties of the two molecular varieties of kallikrein k10 towards synthetic fluorogenic substrates are not significantly different. Both cleave specifically after Arg residues, but, in contrast to true tissue kallikrein, may accommodate either polar or nonpolar residues at position P2. Kallikrein k10 also differs from tissue kallikrein by its sensitivity to soyabean trypsin inhibitor. Its biological function may therefore differ from that of tissue kallikrein, especially as it does not induce a transient decrease in blood pressure when injected in vivo.     

Human prostate-specific antigen (APS) is a member of the glandular kallikrein gene family at 19q13

The amino acid sequence of human prostate-specific antigen (APS) suggests that it is a member of the glandular kallikrein subfamily of serine proteases. In the mouse, the kallikrein-like family is localized in a single locus on chromosome 7, while other serine proteases are distributed over a variety of different chromosomes. To investigate the physical relationship between the human kallikrein genes, we have used in situ hybridization and Southern analysis of a human x mouse somatic cell hybrid panel to map the APS gene to 19q13, concordant with the renal kallikrein KLK1 gene. This finding indicates that APS is a member of a human kallikrein-like gene family with analogous organization to that of the mouse.     

Mouse glandular kallikrein genes. Nucleotide sequence of cloned cDNA coding for a member of the kallikrein arginyl esteropeptidase group of serine proteases

A library of cloned cDNA to male mouse submaxillary gland poly(A)-containing RNA was constructed in the plasmid pBR322. Inserts containing sequences estimated to be in the 1-5% abundance class were identified by hybridization to radiolabeled cDNA and examined by nucleotide sequence analysis. A sequence coding for a peptide with 57% homology to the only complete kallikrein sequence reported to date (from pig pancreas) was identified by a computer search program. This insert appears to code for the COOH-terminal 149 amino acids of a protein presumed therefore to be a serine protease. Comparison of the predicted amino acid sequence of this protein with analogous sequences in the three characterized members of the mouse submaxillary gland kallikrein arginyl esteropeptidase group of enzymes revealed extensive homology, although not complete identity. Thus, there are at least four members of this enzyme family expressed in the mouse submaxillary gland.     

Localization of kallikrein in the guinea-pig submandibular gland

The localization of kallikrein in the acinar and the ductal components of the guinea-pig submandibular gland was investigated by a microdissection technique and an esterase assay. The results indicated that a major portion of the kallikrein is located in the striated duct cells. These results disagree with previous studies in the guinea-pig but are consistent with findings in the rat and cat submandibular gland.     

Tissue kallikrein mK13 is a candidate processing enzyme for the precursor of interleukin-1beta in the submandibular gland of mice

By using Western blot analysis, high levels of 17.5- and 20-kDa interleukin-1beta (IL-1beta) proteins were detected in the submandibular gland (SMG) of mice. Despite this fact, the amount of pro-IL-1beta protein, a precursor of IL-1beta, with a molecular size of 35 kDa in this tissue was below the detectable level, although strong expression of pro-IL-1beta mRNA was observed. A large amount of 17.5-kDa IL-1beta also appeared in the saliva of mice injected with lipopolysaccharide, suggesting that this IL-1beta is a secretory form produced by the SMG. The protein for IL-1beta-converting enzyme, a processing enzyme for pro-IL-1beta, was expressed only at a low level in the SMG as compared with its level in various epithelial tissues or lipopolysaccharide-stimulated macrophages. On the other hand, mK1, mK9, mK13, and mK22, members of the kallikrein family, were detected strongly in the SMG but not in other tissues. By incubation with mK13, but not with mK1, mK9, or mK22, the 35-kDa pro-IL-1beta was cleaved into two major products with molecular masses of 17.5 and 22 kDa, and production was inhibited by phenylmethylsulfonyl fluoride, a serine protease inhibitor, but not by IL-1beta-converting enzyme inhibitors. A peptide segment corresponding to amino acid residues 107-121 of mouse pro-IL-1beta (107WDDDDNLLVCDVPIR) was cleaved by incubation with mK13, generating two peptides, 107WDDDDNL and 114LVCDVPIR. Therefore, kallikrein mK13 would appear to hydrolyze pro-IL-1beta between its Leu113 and Leu114 residues. The results of immunohistochemistry and an autonomic therapy experiment showed that IL-1beta and kallikrein mK13 were co-localized in the secretory granules of granular convoluted tubular cells. Our present results thus suggest kallikrein mK13 is a plausible candidate for the processing enzyme for pro-IL-1beta in the SMG of mice.     

Oestrogen administration and the expression of the kallikrein gene family in the rat submandibular gland

Using a series of oligonucleotide probes (18-21 mers) specific for members of the rat kallikrein/tonin (arginyl-esteropeptidase) gene family (PS, S1, S2, S3, K1, P1), we have shown by Northern blot analysis that all six genes are expressed in the submandibular gland (SMG), with PS (true kallikrein) the most abundant in both male and female rats. Though female levels of PS mRNA are similar to that in the male, levels of mRNA from both the kallikrein-like (S1, K1, P1) and tonin (S2)/tonin-like (S3) genes are all substantially lower in the female than in the male rat. In contrast with the oestrogen dependence of anterior pituitary kallikrein (PS) gene expression, oestrogen administration (6 micrograms/day for 8 days) to castrate male or female rats is without effect on PS or S1, S2, S3, K1, P1 mRNA levels in the SMG. These findings suggest a tissue-specificity in the oestrogen regulation of true kallikrein gene expression in the two tissues. In intact male rats, oestrogen administration lowers SMG levels of S1, S2, S3, K1, and P1 but not PS mRNA to castrate levels, presumably by suppression of the pituitary/gonadal axis, consistent with the previously reported androgen dependence of SMG expression of these genes with the exception of PS.     

Direct angiotensin II formation by rat submandibular gland kallikrein

Submandibular gland kallikrein [EC 3.4.21.8] of male Sprague-Dawley rats was purified by chromatography on soybean trypsin inhibitor (SBTI)-CH-Sepharose 4B, DEAE-Sephadex A-50, aprotinin-CH-Sepharose 4B and Sephadex G-100 columns and preparative isoelectrofocusing. The molecular weight of the kallikrein was estimated to be 30,000 by Sephadex G-100 gel filtration and 29,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric points ranged from pH 3.55 to 4.30. The kinin formed at pH 8 by this kallikrein from bovine low molecular weight (LMW) kininogen showed the same behavior as lysyl-bradykinin on HPLC in a solution of ammonium biphosphate containing acetonitrile. At physiological pH, this kallikrein also generated angiotensin II, a potent vasopressor, from human plasma protein. Rat submandibular gland kallikrein differs from tonin in the isoelectric point, the optimal pH for angiotensin II formation and the type of kinin formed. The tissue kallikrein might play a role in the regulation of local blood flow in view of its ability to form both vasoconstrictive and vasodilatory peptides.     

Mouse glandular kallikrein genes. Structure and partial sequence analysis of the kallikrein gene locus

Mouse glandular kallikreins are encoded by a family of closely linked genes which are located on chromosome 7 at a site corresponding to the genetically defined Tam-1, Prt-4, and Prt-5 loci. We have characterized 24 kallikrein genes by genomic cloning and restriction mapping of 310 kilobase pairs of BALB/c mouse DNA. Most of these genes are highly homologous, have the same exon/intron organization, and are linked in clusters of up to 11 genes. Partial sequence analysis of the kallikrein genes has facilitated identification of those members of the family for which protein sequence data exist and assignment of those which are pseudogenes or encode proteins of unknown function. We find that a maximum of 14 mouse kallikrein genes have the potential to encode functional proteins.     

Single-chain tissue-type plasminogen activator is a substrate of mouse glandular kallikrein 24

Leydig cells of the adult mouse testis express at a detectable level three distinct glandular (tissue) kallikrein genes: mKlk21, mKlk24, and mKlk27. Recently, the proteins encoded by these genes were characterized using active recombinant proteases, but their roles in the mouse testis remained to be determined. The present study showed that among the proteases, mK24 markedly enhanced the activity of human recombinant single-chain tissue-type plasminogen activator when the two were incubated together. This activation was found to be due to proteolytic conversion of the single-chain enzyme to a two-chain form. The expression of tissue-type plasminogen activator in interstitial Leydig cells was demonstrated by RT-PCR and immunohistochemical analyses. The primary culture medium of adult male testicular Leydig cells contained immunoreactive substances recognized by anti-mK24 antibodies. In addition, the same medium was capable of converting the single-chain plasminogen activator to the two-chain protein. These results suggest that mK24 may play a role in the degradation of extracellular matrix proteins in the interstitial area surrounding the Leydig cells of the adult mouse testis, due not only to its own activity, but also to that of plasmin produced by the single-chain tissue-type plasminogen activator-converting activity of mK24.     

A simple and rapid purification of kallikrein from rat submandibular gland.

Rat submandibular kallikrein was isolated in an 87% yield by a very quick and simple procedure involving hydrophobic interaction chromatography. Furthermore, that purification method was superior to both aprotinin-affinity chromatography and immunoaffinity chromatography for the purification of rat submandibular kallikrein. The kallikrein purified by hydrophobic interaction chromatography consisted of a number of isoenzymes. The major component of Mr 38,000 seen on SDS-gel electrophoresis was found to be the glycosylated kallikrein, whereas the minor component of Mr 26,000 represented the non-glycosylated enzyme.     

Mouse oncogene protein 24p3 is a member of the lipocalin protein family.

Rigorous new methods of protein sequence analysis have been applied to the lipocalins, a diverse family of ligand binding proteins. Using three conserved sequence motifs to search for similar patterns in a large sequence database, the size and composition of this protein family have been defined in an automatic and objective way. It has allowed the identification of an existing sequence, mouse 24p3 protein, as a lipocalin and the possible rejection of other putative members from this protein family. On the basis of this newly discovered homology, a possible function for mouse 24p3 protein is proposed.     

The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases

We recently demonstrated that the major decapping activity in mammalian cells involves DcpS, a scavenger pyrophosphatase that hydrolyzes the residual cap structure following 3' to 5' decay of an mRNA. The association of DcpS with 3' to 5' exonuclease exosome components suggests that these two activities are linked and there is a coupled exonucleolytic decay-dependent decapping pathway. We purified DcpS from mammalian cells and identified the cDNA encoding a novel 40 kDa protein possessing DcpS activity. Consistent with purified DcpS, the recombinant protein specifically hydrolyzed methylated cap analog but did not hydrolyze unmethylated cap analog nor did it function on intact capped RNA. Sequence alignments of DcpS from different organisms revealed the presence of a conserved hexapeptide, containing a histidine triad (HIT) sequence with three histidines separated by hydrophobic residues. Mutagenesis analysis revealed that the central histidine within the DcpS HIT motif is critical for decapping activity and defines the HIT motif as a new mRNA decapping domain, making DcpS the first member of the HIT family of proteins with a defined biological function.     

Mouse whey acidic protein is a novel member of the family of 'four-disulfide core' proteins.

Unlike in other mammalian species, the major whey protein in mouse is not alpha-lactalbumin, but a cysteine rich, acidic protein with a molecular weight of 14.0 kDa. We have deduced the amino acid sequence of this mouse acidic of whey protein from the nucleotide sequence of cloned cDNA. The positions of the half cysteines suggest that mouse whey acidic protein (WAP) is a two domain protein, very similar in structure to the plant lectin wheat germ agglutinin and the hypothalamic carrier protein neurophysin.     

The aging-associated enzyme CLK-1 is a member of the carboxylate-bridged diiron family of proteins

The aging-associated enzyme CLK-1 is proposed to be a member of the carboxylate-bridged diiron family of proteins. To evaluate this hypothesis and characterize the protein, we expressed soluble mouse CLK-1 (MCLK1) in Escherichia coli as a heterologous host. Using Mo?ssbauer and EPR spectroscopy, we established that MCLK1 indeed belongs to this protein family. Biochemical analyses of the in vitro activity of MCLK1 with quinone substrates revealed that NADH can serve directly as a reductant for catalytic activation of dioxygen and substrate oxidation by the enzyme, with no requirement for an additional reductase protein component. The direct reaction of NADH with a diiron-containing oxidase enzyme has not previously been encountered for any member of the protein superfamily.     

The relationship between glandular kallikrein and growth factor-processing proteases of mouse submaxillary gland.

Three highly specific trypsin-like proteases from mouse submaxillary gland; nerve growth factor gamma subunit, beta nerve growth factor-endopeptidase, and epidermal growth factor-binding protein were tested for kallikrein activity. Low molecular weight kininogen was purified from mouse plasma and used as substrate for the three enzymes, and the kinin released by the enzymes was assayed by its ability to induce contraction of isolated rat uterus. All three enzymes were found to have significant kininogenase activity, and the most active enzyme, beta nerve growth factor-endopeptidase, has activity comparable to authentic kallikreins from other glandular sources. Essentially all of the kininogenase activity of submaxillary gland co-purifies with beta nerve growth factor-endopeptidase. Hence, beta nerve growth factor-endopeptidase appears to be identical with submaxillary gland kallikrein. Nerve growth factor gamma subunit, epidermal growth factor-binding protein, and beta nerve growth factor-endopeptidase have similar amino acid compositions and molecular weights, and are immunologically similar. Comparison of published partial primary sequence data confirms our conclusion that nerve growth factor gamma subunit, epidermal growth factor-binding protein, and kallikrein are very closely related enzymes. It is postulated that these three enzymes are members of a larger family of similar enzymes, all of which are involved in the processing of precursors to polypeptide hormones and growth factors.     

Comparison of genetic and endocrine control of renin and kallikrein in mouse submandibular gland

The effects of strain, sex, hypophysectomy and hormone treatment on mouse submandibular gland renin, kallikrein, S2266 hydrolase, and BAEe esterase activities have been examined. Renin activity is determined by the $\text{Rnr}$ locus on mouse Chromosome 1. Female SWR/J mice ($\text{Rnr}$^$\text{s}$/$\text{Rnr}$^$\text{s}$) have 1000-fold higher submandibular gland renin activity than C57BL/6J mice ($\text{Rnr}$^$\text{b}$/$\text{Rnr}$^$\text{b}$). Both strains have similar kallikrein activity. Renin, BAEe esterase, and S2266 hydrolase are substantially higher in male mice compared to females of the same strain whereas kallikrein is not. Dihydrotestosterone and/or thyroxine treatment induces renin, BAEe esterase, and S2266 hydrolase in female mice with little effect on kallikrein. All four enzyme activities are profoundly reduced by hypophysectomy. Dihydrotestosterone and thyroxine are both required to restore renin, BAEe esterase, and S2266 hydrolase to induced levels. Dihydrotestosterone and.or thyroxine restores kallikrein to control levels. We conclude that renin and kallikrein in the mouse submandibular gland are under different genetic and endocrine control. In addition, the synthetic substrate S2266 is not a specific substrate for kallikrein activity in mouse submandibular gland cytosol.