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T-kininogenase activity of the rat submandibular gland is predominantly due to the kallikrein-like serine protease antigen gamma.

ABSTRACT: T-kininogen, the major kininogen in rat plasma, releases Ile-Ser-bradykinin (T-kinin) when incubated with trypsin, but is not a substrate for tissue kallikrein. Enzymes able to release T-kinins from T-kininogen have been found in the rat submandibular gland, but precise identification of these enzymes and their possible relationship to kallikrein-like enzymes has not been established. We studied T-kininogenase activity in fractionated submandibular gland homogenate. The main T-kininogen catalytic enzyme was purified and characterized, and found to be identical to antigen gamma, a kallikrein-like enzyme which we have previously characterized. Of other identified kallikrein-like enzymes only tonin showed weak T-kininogenase activity, which was about 0.25% of that of antigen gamma. No other T-kininogen catalytic enzymes were observed. Antigen gamma released a kinin which was identified as T-kinin by reverse-phase h.p.l.c. The T-kininogenase activity of antigen gamma had a Km of 29 +/- 4 microM and a kcat/Km of 140 M-1.s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10 micrograms of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42,200 micrograms of kinin/h per mg respectively. Thus antigen gamma is a weak kininogenase. The isoelectric point of antigen gamma, but not its molecular mass, differed from that of other kallikrein-like enzymes. Isoelectrofocusing in flat-bed gels combined with immunostaining was therefore a convenient method for identification. The kallikrein-like nature of antigen gamma was demonstrated by its immunological similarity to tissue kallikrein and tonin and by 91% and 87% amino acid sequence similarity with tonin and kallikrein respectively (67 amino acids sequenced). Complete identity was also not observed with other sequenced kallikrein genes, mRNAs or proteins.

SUBMITTER: Berg T

PROVIDER: S-EPMC1130593 | biostudies-other | 1991 Nov

REPOSITORIES: biostudies-other

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Project description:The submandibular gland of the rat contains several enzymes belonging to the kallikrein family. These include tissue kallikrein, antigen gamma (T-kininogenase), esterase B and tonin. In the present study, a new member of this family, which we have named KLP-S3, was identified and purified from the submandibular gland. KLP-S3 was classified as a kallikrein-like enzyme on the basis of its immunological similarity to other kallikrein-like enzymes and its showing 70% and 73% identity in partial amino acid sequence with tissue kallikrein and tonin respectively. Furthermore, the 44 sequenced amino acid residues showed complete correspondence to the mRNA S3 of the kallikrein gene family, which was the rationale for the name kallikrein-like protein (KLP) S3. KLP-S3 consisted of three isoenzymes with pI 6.75, 6.90 and 6.95, which significantly differed from those of other kallikrein-like enzymes. In conjunction with its immunological relationship to kallikrein, this parameter (pI) was considered robust enough to identify the enzyme during purification, since a specific physiological substrate for KLP-S3 has yet to be identified. In SDS/PAGE the three isoenzymes ran as one band with a molecular mass of 25,800 Da, which after reduction with 2-mercaptoethanol was split into two chains with molecular masses of 16,500 and 13,300 Da. In common with other kallikrein-like enzymes, KLP-S3 was inhibited by phenylmethanesulphonyl fluoride, and was thus classified as a serine protease. It was also inhibited by soya-bean trypsin inhibitor but not by aprotinin. It showed weak reactivity against the chromogenic substrates S2288, S2266, S2366 and S2302 (D-Ile-Pro-Arg 4-nitroanilide, D-Val-Leu-Arg 4-nitroanilide, Glu-Pro-Arg 4-nitroanilide and D-Pro-Phe-Arg 4-nitroanilide respectively) and did not cleave rat T-kininogen or dog high-molecular-mass/low-molecular-mass kininogen. Its specific angiotensin II-generating activity (angiotensin I as substrate) was 0.04% of that of rat tonin. KLP-S3 (1-100 nM) induced a statistically significant angiotensin-independent contraction of isolated rat aorta rings. The maximum contraction was 15% of the response to the alpha-adrenoceptor agonist phenylephrine (1 microM). The concentration of KLP-S3 in the rat submandibular gland was by single radial immunodiffusion estimated to be 47 +/- 3 micrograms/mg of protein.

Project description:BackgroundOrgan replacement regenerative therapy based on human adult stem cells may be effective for salivary gland hypofunction. However, the generated tissues are immature because the signaling factors that induce the differentiation of human salivary gland stem cells into salivary glands are unknown.MethodsIsolated human submandibular gland stem/progenitor cells (hSMGepiS/PCs) were characterized and three-dimensionally (3D) cultured to generate organoids and further induced by fibroblast growth factor 10 (FGF10) in vitro. The induced spheres alone or in combination with embryonic day 12.5 (E12.5) mouse salivary gland mesenchyme were transplanted into the renal capsules of nude mice to assess their development in vivo. Immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, calcium release analysis, western blotting, hematoxylin-eosin staining, Alcian blue-periodic acid-Schiff staining, and Masson's trichrome staining were performed to assess the structure and function of generated tissues in vitro and in vivo.ResultsThe isolated hSMGepiS/PCs could be long-term cultured with a stable genome. The organoids treated with FGF10 [FGF10 (+) group] exhibited higher expression of salivary gland-specific markers; showed spatial arrangement of AQP5+, K19+, and SMA+ cells; and were more sensitive to the stimulation by neurotransmitters than untreated organoids [FGF10 (-) group]. After heterotopic transplantation, the induced cell spheres combined with mouse embryonic salivary gland mesenchyme showed characteristics of mature salivary glands, including a natural morphology and saliva secretion.ConclusionFGF10 promoted the development of the hSMGepiS/PC-derived salivary gland organoids by the expression of differentiation markers, structure formation, and response to neurotransmitters in vitro. Moreover, the hSMGepiS/PCs responded to the niche in mouse embryonic mesenchyme and further differentiated into salivary gland tissues with mature characteristics. Our study provides a foundation for the regenerative therapy of salivary gland diseases.

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T-kininogenase activity of the rat submandibular gland is predominantly due to the kallikrein-like serine protease antigen gamma.

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