Lee Chao

Tissue distribution and kininogen gene expression after acute-phase inflammation.

A kinin-directed monoclonal antibody to kininogens has been developed by the fusion of murine myeloma cells with mouse splenocytes immunized with bradykinin-conjugated hemocyanin. The hybrid cells were screened by an enzyme-linked immunosorbent assay (ELISA) and a radioimmunoassay (RIA) for the secretion of antibodies to bradykinin. Ascitic fluids were produced and purified by a bradykinin-agarose affinity column. The monoclonal antibody (IgG1) bound to bradykinin, Lys-bradykinin, Met-Lys-bradykinin, and kininogens in ELISA. Further, this target-directed monoclonal antibody recognized purified low and high molecular weight bovine, human, or rat kininogens and T-kininogen in Western blotting. After turpentine-induced acute inflammation, rat kininogen levels increased dramatically in liver and serum as well as in the perfused pituitary, heart, lung, kidney, thymus, and other tissues, as identified by the kinin-directed kininogen antibody in Western blot analyses. The results were confirmed by measuring kinin equivalents of kininogens with a kinin RIA. During an induced inflammatory response, rat kininogens were localized immunohistochemically with the kinin-directed monoclonal antibody in parenchymal cells of liver, in acinar cells and some granular convoluted tubules of submandibular gland, and in the collecting tubules of kidney. Northern and cytoplasmic dot blot analyses using a kinin oligonucleotide probe showed that kininogen mRNA levels in liver but not in other tissues increase after turpentine-induced inflammation. The results indicated that rat kininogens are distributed in various tissues in addition to liver and only liver kininogen is induced by acute inflammation. The target-directed kininogen monoclonal antibody is a useful reagent for studying the structure, localization, and function of kininogens or any protein molecule containing the kinin moiety.

Restriction fragment length polymorphisms mapped in spontaneously hypertensive rats using kallikrein probes

We have explored the role of kallikrein-kinin system in essential hypertension using spontaneously hypertensive rats (SHR) as an animal model. A rat tissue kallikrein complementary (c) DNA (RSK 1105) was used as a probe in Southern blot hybridization to detect restriction fragment length polymorphisms (RFLPs) in SHR. Using 23 different restriction endonucleases, we have identified five RFLPs involving alterations in restriction fragment lengths for the restriction enzymes Bgl II, Dra I, Nde I, Sph I, and Bcl I. Three of the enzymes, Nde I, Sph I, and Bgl II, generate multiple polymorphic fragments. We have further mapped these RFLPs with two additional probes, both from the rat renal kallikrein gene RSKG 7. The 5 probe, consisting of sequences approximately 2000 base pair (bp) 5 of the first exon, recognizes RFLPs in DNA digested with Bcl I and Sph I. The 3 probe, approximately 4400 bp away from the fifth exon, recognizes polymorphic fragments in DNA digested with Bcl I, Dra I and Nde I. These findings indicate possible differences in tissue kallikrein genes or their regulatory regions in SHR that could contribute to the pathogenesis of hypertension in this animal model.

Sex dimorphism and hormonal regulation of rat tissue kallikrein mRNA

The regulation of rat tissue kallikrein mRNA levels was investigated by RNA filter hybridization using a kallikrein cDNA probe and by in vitro translation of isolated RNA. The synthesis of a 37 kDa polypeptide has been hybrid-arrested by the tissue kallikrein cDNA in cell-free translation assays directed by submandibular mRNA and the quantitation of this protein was used as a measure of translational activity of kallikrein mRNA. In Northern blot analyses, relative kallikrein mRNA levels were found to be 60-fold higher in submandibular gland than in pancreas and 20-fold higher than in kidney. In addition, it was noted that kallikrein mRNA is differentially regulated in tissues from male and female animals. Rat submandibular gland kallikrein mRNA is 2-times more abundant in male animals as in females. This trend is reversed in kidney, where the kallikrein mRNA in females is twice that in male animals. Relative submandibular gland kallikrein mRNA levels were found to be responsive to hormonal manipulation as measured by both Northern blotting and cell-free translation assays. Castration of male animals resulted in a decrease of kallikrein mRNA which could be partially restored by treatment with thyroxine and almost completely restored by testosterone treatment. The submandibular gland kallikrein mRNA in normal female rats was increased up to 30% by administration of testosterone or thyroxine. Adrenalectomized female rats showed a decrease in submandibular kallikrein mRNA which was significantly increased by testosterone, thyroxine, aldosterone and cortisol, with the largest effect being a 2- to 2.4-fold increase in cortisol-treated rats. These results indicate that hormones regulate tissue kallikrein synthesis in the rat submandibular gland, at least in part, by altering kallikrein mRNA accumulation.

Isolation of tissue kallikrein in rat spleen by monoclonal antibody-affinity chromatography

Rat spleen kallikrein was identified and purified by DEAE-cellulose and monoclonal antibody-affinity chromatography. The purified enzyme has Tos-Arg-OMe esterase activity and kinin-releasing activity from a purified low-molecular-weight kininogen substrate. In the direct radioimmunoassay for tissue kallikrein, the splenic enzyme displays parallelism with standard curves of rat urinary kallikrein. The pH profiles of the Tos-Arg-OMe esterase activities of spleen and urinary kallikrein were identical with optima at 9.0. Rat spleen kallikrein was inhibited strongly by aprotinin and affinity-purified kallikrein antibody and weakly by soybean trypsin inhibitor. The IC50 values were similar to those observed against rat urinary kallikrein. Neither the urinary nor the splenic enzyme was inhibited by lima bean trypsin inhibitor or preimmune serum immunoglobulins. Spleen kallikrein was labeled with [14C]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. The electrophoretic mobility of the splenic enzyme was indistinguishable from that of urinary kallikrein A with an estimated Mr of approx. 38 000. With Western blot analyses using a rabbit anti-kallikrein antibody followed by 125I-labeled protein A binding, the spleen and urinary kallikreins were again visualized at identical positions by autoradiography. The data show that there is a rat splenic tissue kallikrein which is indistinguishable from a renal kallikrein with respect to physicochemical properties, immunological character and susceptibility to inhibitors.

Identification and expression of kallikrein gene family in rat submandibular and prostate glands using monoclonal antibodies as specific probes

Panels of monoclonal antibodies to three vasoactive peptide-producing enzymes: tissue kallikrein, tonin and arginine esterase A were developed, characterized and used as probes for identification of tissue-specific expression. In addition, immunoblot analyses were performed, using monospecific monoclonal antibodies which did not show cross-reactivity to related-purified enzymes in enzyme-linked immunosorbant assay (ELISA), and radioimmunoassay. We obtained the following results. In rat submandibular gland extract, the expression of 38 kDa kallikrein, 32 kDa tonin, and 18 kDa heavy chain of esterase A was identified by monoclonal antibodies to kallikrein (V4D11), tonin (1F11), and esterase A (5A10, 6C11, and 4B12), respectively. In the prostate gland, a 32 kDa kallikrein-like protein was identified by monoclonal antibodies to esterase A (5A10, 6C11 and 4B12) and by antibodies recognizing both tonin and esterase A (5A5), but not by antibody to kallikrein (V4D11) or to tonin (1F11, 1G6) in Western blot analysis. The esterase A-like enzyme in the prostate gland was found within the cytoplasm of ductal epithelial cells by using monoclonal anti-esterase A antibody (5A10) but not by employing anti-tonin antibody (1F11). These results indicate that tissue kallikrein, tonin, and esterase A are all expressed in the submandibular gland, while only esterase A or an esterase A-like enzyme is expressed in the prostate gland. The specific monoclonal antibodies can be used as probes for the identification and expression of the kallikrein gene-family enzymes.

Sex dimorphism and inflammatory regulation of T-kininogen and T-kininogenase

Studies were carried out in order to better understand hormonal and inflammatory regulation of the T-kininogen and T-kininogenase system. T-kininogen from rat serum and T-kininogenase from rat submandibular gland were purified to homogeneity, and specific antisera to the purified proteins were generated. Simple, sensitive and specific radioimmunoassays were developed for measuring both T-kininogen and T-kininogenase. The assays incorporated a modified poly(ethylene glycol) technique for separating free from antibody-bound forms. Optimal combinations of poly(ethylene glycol) and gamma-globulin were found, yielding low background and high specific binding. The assays can detect a minimum of 160 pg of T-kininogen and 80 pg of T-kininogenase per tube. Serial dilutions of sera from normal and turpentine-treated rats showed complete parallelism with the T-kininogen standard curve. T-kininogen levels in rat serum and rat tissues increased more than 10-fold following turpentine treatment, while T-kininogenase levels in the submandibular gland and other tissues remained unchanged. Through use of a kinin-directed kininogen monoclonal antibody, Western blots of two-dimensional gels of serum following acute inflammation showed increased levels of several kininogens which vary in both molecular weight and isoelectric point. Analysis of serum kininogen levels shows sexual dimorphism, with female rats having 3.9-fold higher levels than males. Contrarily, T-kininogenase levels in the submandibular gland of male rats are 2.4-fold higher than those in females. The studies also showed that the T-kininogen and T-kininogenase system is regulated by sex hormones. T-kininogen is an acute-phase protein whose rapid increase and mobilization following inflammation may provide a primary defense against proteolytic damage during trauma.

Identification of a kallikrein-like latent serine protease in human erythrocyte membranes

We have discovered and characterized a kallikrein-like latent serine protease in intact human erythrocytes and ghosts. The enzyme is activatable by trypsin. The solubilized enzyme has esterolytic activity with a pH optimum of 9; but the membrane-associated activity increases almost linearly up to pH 10. The activated enzyme releases kinin from bovine low molecular weight kininogen. Enzyme activity is inhibited by TosLysCH2Cl , phenylmethylsulfonyl fluoride, aprotinin and amiloride, and weakly by soybean or lima bean trypsin inhibitor. It is inhibited by Co2+, Zn2+ and Mn2+ but is stimulated by Fe2+, deoxycholate and phospholipase A2. An erythrocyte membrane protein (Mr = 88,000) with an active site serine residue was identified with [14C]-diisopropylphosphorofluoridate labeling. Consistent with the finding of tryptic activation of the latent erythrocyte serine protease, trypsin treatment reduced the density of labeling of this protein and revealed a lower molecular weight form (Mr = 64,000). Possible relationships between the activity of this newly identified serine protease and events such as erythrocyte membrane ion fluxes might be of interest.

Immunological analysis of rat pancreatic prokallikrein activation

The present study shows that tissue kallikrein is present in rat pancreas as a proenzyme that can be converted by autolysis to a 38 000 Da active enzyme. The activation of pancreatic prokallikrein was examined by direct radioimmunoassay, enzymatic assays, active-site labeling with immunoprecipitation, and Western blot analyses. A monoclonal antibody (V1C3), which binds only active kallikrein, was used in a direct radioimmunoassay to monitor the appearance of the active enzyme. During a 22-h autolysis of pancreatic extract, a time-dependent increase in active kallikrein concentration paralleled the increase of kallikrein activities measured by both TosArgOMe esterase and kininogenase assays. The activation process was further analyzed by labeling the pancreatic extract with [14C]diisopropylphosphorofluoridate [( 14C]DFP) followed by immunoprecipitation with sheep anti-kallikrein antiserum. Pancreatic prokallikrein was not labeled by [14C]DFP; however, upon autolysis, a 38 000 Da active kallikrein can be labeled with [14C]DFP and increase in quantity with time. Western blot analysis, using a monoclonal antibody (V4D11) which recognizes both latent and active tissue kallikreins, identified a 39 000 Da pancreatic prokallikrein prior to autolysis and a 38 000 Da active kallikrein after 7 h of autolysis. The results indicate that the pancreatic prokallikrein exists as a 39 000 Da protein which may be converted to a 38 000 Da active kallikrein, indistinguishable from purified urinary, brain, spleen or submandibular gland kallikrein.

Active Kallikrein, Preprokallikrein, and Kallikrein-Inhibitor Complex

Active kallikreins isolated from various exocrine and endocrine tissues were identified by a monoclonal antibody in Western blot analyses to be approximately 38,000 dalton proteins. Kallikreins isolated from rat pancreas, kidney, submandibular gland, brain, spleen and urine were indistinguishable with respect to molecular weight and immunological characteristics. Preprokallikreins were synthesized in a cell-free translation system directed by mRNAs and immunoprecipitated by affinity-purified kallikrein antibody. Analysis of the precipitates by SDS-polyacrylamide gel electrophoresis revealed a approximately 37,000 dalton polypeptide in kidney, brain and submandibular gland translation products. This 37,000 dalton kallikrein precursor was hybrid-arrested by a kallikrein cDNA encoding tissue kallikrein which was isolated from a rat submandibular gland cDNA library. The immunoprecipitates of products directed by pancreatic mRNA showed a major protein with Mr of approximately 30,000. An endogenous approximately 92,000 dalton component in rat urine and kidney was also identified by a monoclonal antibody to tissue kallikrein and represents a kallikrein-inhibitor complex. These results indicate that tissue kallikreins can be initially synthesized as 37,000 or 30,000 dalton prepropeptides and then converted into a 38,000 dalton active form by proteolytic processing and glycosylation. The active kallikrein is capable of binding to an inhibitor to form a 92,000 dalton complex.

Characterization of Rat Kallikrein-Like Multigene Family and its Expression in the Submandibular Gland

A cDNA clone encoding rat tissue kallikrein was isolated from a submandibular cDNA library. The kallikrein cDNA clone was used as a probe to analyze the complexity of the kallikrein-like gene family and its expression. The results indicate that rat kallikrein-like genes identified with this probe belong to a very large and highly homologous multigene family. A number of these genes, perhaps as many as a dozen or so, are expressed in the submandibular gland.