Kininogen-derived fluorogenic substrates for investigating the vasoactive properties of rat tissue kallikreins--identification of a T-kinin-releasing rat kallikrein.

Peptide substrates with intramolecularly quenched fluorescence that reproduce the rat kininogen sequences at both ends of the bradykinin moiety were synthesized and used to investigate the kinin-releasing properties of five rat tissue kallikreins (rK1, rK2, rK7, rK9, rK10). Substrates derived from rat H- and L-kininogen were cleaved best by rK1, especially that including the N-terminal insertion site of bradykinin, Abz-TSVIRRPQ-EDDnp(Abz = O-aminobenzoyl, EDDnp = ethylenediamine 2,4-dinitrophenyl), which was cleaved at the R-R bond with a k(cat)/Km of 12400 mM(-1) s(-1). Replacement of the P2' residue Pro by Val in Abz-TSVIRRPQ-EDDnp gave a far less specific substrate that was rapidly hydrolysed by all five rat kallikreins and human kallikrein hK1. Peptidyl-N-methyl coumarylamide substrates, which lack prime residues, also had low specificities. The importance of the P2' residue for rK1 specificity was further demonstrated using a human-kininogen-derived substrate that included the N-terminal insertion site of bradykinin (Abz-LMKRP-EDDnp). This was cleaved at the M-K bond by hK1 (kallidin-releasing site), but at the K-R bond (bradykinin-releasing site) by rK1. Competition experiments with Abz-TSVIRRPQ-EDDnp, which is resistant to most kallikreins, and Abz-TSVIRRVQ-EDDnp, a general kallikrein substrate, demonstrated that the former competitively inhibited hydrolysis by rK9 and hK1, with Ki values similar to the Km values for the substrate. Thus Pro in P2' does not prevent the peptide binding to the enzyme active site, but impairs cleavage of the scissile bond. The T-kininogen-derived substrate with the T-kinin C-terminal sequence (Abz-FRLVR-EDDnp) was cleaved by rK10 (k(cat)/Km = 2310 mM(-1) s(-1)) and less rapidly by rK1, rK7 and hK1, at the R-L bond, while that corresponding to the N-terminal (Abz-ALDMMISRP-EDDnp) of T-kinin was resistant to all five kallikreins used, suggesting that none has T-kininogenase activity. But this substrate was hydrolysed by a semipurified sample of submandibular gland extract. Another kallikrein, identified as kallikrein rK3, was isolated from this fraction and shown to hydrolyze Abz-ALDMMISRP-EDDnp; rK3 also specifically released T-kinin from purified T1/T2-kininogen after HPLC fractionation. Injection of purified rK3 and of Abz-ALDMMISRP-EDDnp-cleaving fractions into the circulation of anesthesized rats caused transient falls in blood pressure, as did purified rK1 but none of the other purified rat or human kallikreins. This effect occurred via activation of the kinin system since it was blocked by Hoe140, a kinin receptor antagonist.

Functional diversity of proteinases encoded by genes of the rat tissue kallikrein family.

A group of proteinases closely related to tissue kallikrein was purified from the rat submandibular gland. Physicochemical characterization of these proteinases, including amino terminal sequencing, allowed correlation with the genes of the rat kallikrein family. In spite of their similar structure, these proteinases have different substrate specificities and different susceptibilities to inhibitors which suggest that they do not share the same biological function. Kallikrein-like proteinases also have restricted specificities that are probably related to their extended substrate binding site. This makes them good candidates for processing inactive protein or peptide precursors into biologically active peptides. A general approach to identifying the putative biological substrates of individual proteinases based on analysis of the specific cleavage of synthetic and natural peptide substrates by kallikrein-related proteinases is described.

Differential rates of glycoprotein secretion by isolated rat hepatocytes studied in terms of concanavalin A binding.

Using a concanavalin-A-based method which respects cell function, we have shown that the kinetics of glycoprotein secretion appear to depend on the nature of the oligosaccharide moiety. In 37 degrees C pulse/chase experiments using freshly isolated normal rat hepatocytes, we found that except for transferrin, whose rate of secretion was independent of its concanavalin A reactivity, the secretion of the concanavalin-A-retained forms of alpha 1 acid glycoprotein, T-kininogen, alpha 1 protease inhibitor and alpha 1 inhibitor III was slower than that of the concanavalin-A-non-retained forms. When hepatocytes were incubated at 20 degrees C, secretion was blocked with the accumulation of mainly endoglycosidase-H-sensitive forms. The secretion kinetics of the concanavalin-A-differentiated forms were still different when the temperature was shifted back to 37 degrees C. The divergence between the secretion rates of the concanavalin-A-differentiated forms would appear to be due to a late event in intracellular protein trafficking, which may depend on the sugar content and/or the number of carbohydrate chains of the glycoproteins.

Possible relationship between the restricted biological function of rat T kininogen (thiostatin) and its behaviour as an acute phase reactant.

Studies on biological properties of rat T kininogen have shown that the role of this peculiar kininogen so far specific to the rat probably differs significantly from that of other low molecular mass kininogens. In particular the kinin precursor function has been either lost or considerably reduced as a result of structural modifications during evolution. The calpain inhibiting function demonstrated for other low and high molecular mass kininogens has also probably disappeared from T kininogen, and since T genes do not allow the synthesis of high molecular mass kininogens [Kitagawa et al. (1987) J. Biol. Chem. 262, 2190-2198], the procoagulant function devoted to the light chain of high molecular mass kininogen has also been lost by T genes products. The only remaining function of rat T kininogen would be therefore that of a lysosomal cysteine proteinase inhibitor which is expressed either by the native molecule or by proteolytic products which appear to be more easily released than vasoactive peptides. Such a specialization for a given function could be related to the behaviour of T kininogen as an acute phase reactant, the dramatic changes in concentration of which could at the same time serve certain functions and be damageable for others.

T-kinin release from T-kininogen by rat-submaxillary-gland endopeptidase K.

Submaxillary gland extracts have been fractionated to characterize the enzyme responsible for the T-kininogenase activity previously reported in this tissue [Damas, J. & Adam, A. (1985) Mol. Physiol 8, 307-316] and to know whether this activity could be of physiological relevance, since no enzyme reacting in catalytic amounts has been described so far to be able to release a vasoactive peptide from T-kininogen. The purified enzyme, provisionally called endopeptidase K, has an apparent Mr of 27,000 when not reduced prior to analysis but 21,000 after reduction and an acidic pI of 4.3 +/- 0.1. Antigenically, it is not related to tissue kallikrein. Upon incubation with purified T-kininogen it may induce a complete liberation of T-kinin from the precursor provided it is added in stoichiometric amounts. However, in parallel with the liberation of immunoreactive kinin, a proteolysis of T-kininogen is observed which is not restricted to the site of insertion of T-kinin as would be expected using a specific kininogenase. In agreement with these results, no change of the mean blood pressure was observed upon injection of endopeptidase K into the circulation of normal rats even if the amount of injected enzyme was up to ten times that required for tissue kallikrein to induce a significant fall in blood pressure. However, in spite of the large proteolysis induced by incubation with stoichiometric amounts of endopeptidase K, the total papain inhibiting capacity of T-kininogen as well as the value of the apparent inhibition constant, Ki, with this proteinase remained unchanged. Proteolytic fragments which retain cysteine-proteinase-inhibiting activity may therefore be released from T-kininogen by endopeptidase K more easily than immunoreactive kinin, thus emphasizing a prominent function of proteinase inhibitor or of proteinase inhibitor precursor for this molecule.

Relationship between the cysteine-proteinase-inhibitory function of rat T kininogen and the release of immunoreactive kinin upon trypsin treatment.

The potential kininogenic function of rat T kininogen has been studied in parallel with the cysteine-proteinase-inhibitory function also carried by this molecule. Proteolytic cleavage of the molecule was observed upon incubation with catalytic amounts of trypsin. These conditions do not permit any significant release of immunoreactive kinin and do not modify the total papain-inhibiting capacity of T kininogen. As trypsin concentration increases in the reaction mixture, immunoreactive kinin is liberated and the total papain-inhibiting capacity decreases accordingly, as indicated by titration studies. This decrease, however, does not exceed 50% of the initial value even at a trypsin concentration as high as 75 microM, indicating that only one of the two inhibitory sites has been inactivated. The remaining inhibitory fragment corresponds to a peptide of apparent Mr 24 000, which binds papain at least as well as native T kininogen. T kininogen, therefore, appears as a potent proteinase inhibitor and/or a proteinase inhibitor precursor, whereas its kininogenic function remains questionable since no specific kininogenase able to release T kinin or another kinin under physiologically compatible conditions has been found so far.

Rat plasma alpha 1-inhibitor3: a member of the alpha-macroglobulin family.

The overall mechanism of interaction with proteinases of alpha 1-inhibitor3, a plasma proteinase inhibitor so far specific to the rat, has been shown to be closely similar to that described for alpha-macroglobulins. This mechanism includes: (i) the cleavage of at least one susceptible peptidic bond which leads to structural changes in the molecule. (ii) The cleavage of a putative thiol ester bond in another site of the molecule which permits the covalent linkage of the enzyme. Moreover, fragmentation of alpha 1-inhibitor3 upon heating as observed for alpha-macroglobulin quarter subunits has been demonstrated. The question is raised of the presence of such a molecule in rat plasma in addition to two alpha-macroglobulin species, all of these proteinase inhibitors being antigenically unrelated.