Protein products of the rat kallikrein gene family. Substrate specificities of kallikrein rK2 (tonin) and kallikrein rK9.

T Moreau,M Brillard-Bourdet,J Bouhnik,F Gauthier

doi:10.1016/s0021-9258(19)50197-1

Abstract

Two closely related kallikrein-like proteinases having little activity toward the standard synthetic amide substrates of tissue kallikreins were isolated from the rat submandibular gland. They were found to be the protein products of the rKlk2 (tonin) and the rKlk9 genes by amino acid sequence analysis (nomenclature of the genes and proteins of the kallikrein family is according to the proposal of the discussion panel from the participants of the KININ '91 meeting held Sept. 8-14, 1991, in Munich, Germany). These two proteinases of similar structure also had very similar physicochemical properties. They differed from other kallikrein-related proteinases in having high pHi values of 6.20 (rK2) and 6.85 (rK9). Kallikrein rK2 was purified as a single peptide chain, whereas rK9 appeared as a two-chain protein after reduction. Their enzymatic properties were also very similar and differed significantly from those of other rat kallikrein-related proteinases. Unlike the five other kallikrein-related proteinases we have purified so far, kallikrein rK9 was not inhibited by aprotinin. rK9 also differed from rK2 by its tissue localization. The prostate gland contained only rK9 where it was the major kallikrein-like component. The amino acids preferentially accommodated by the proteinase S3 to S2' subsites were identified using synthetic amide and protein substrates. Unlike other kallikrein-related proteinases, rK2 had a prevalent chymotrypsin-like specificity, whereas rK9 had both chymotrypsin-like and trypsin-like properties. Both rK2 and rK9 preferred a prolyl residue in position P2 of the substrate and did not accommodate bulky and hydrophobic residues at that position, as did most of the other kallikrein-related proteinases. This P2-proline-directed specificity is necessary for processing the precursors of several biologically active peptides. Subsites accommodating residues COOH-terminal to the scissile bond were also important in determining the overall substrate specificity of these proteinases. rK2 and rK9 both showed a preference for hydrophobic residues in P2'. Other subsites upstream of the S3 subsite were found to intervene in substrate binding and hydrolysis. The restricted specificity of rK2 and rK9 is consistent with the presence of an extended substrate binding site, and hence with a processing enzyme function. Their P1 specificities enabled both proteinases to release angiotensin II from angiotensinogen and from angiotensinogen I, but rK9 was at least 100 times less active than rK2 on both substrates. The substrate specificities of rK2 and rK9 were correlated with key amino acids defining their substrate binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Highlights

Two closely related kallikrein-like proteinases hav- enzyme function
Tissue kallikrein’ and tonin, encoded by the r K 9 was not inhibited by aprotinin. r K 9 differed genes rKlkl and rKlk2, from rK2by its tissue localization
Tion relationships in this proteinase family. We have determined their overall substrate specificityusing synthetic amide and ester substrates as well as natural peptide and protein substrates. This specificity has been tentatively correlated with differences in the key amino acids which define the substrate binding site in the members of the kallikrein family

Summary

Introduction

Two closely related kallikrein-like proteinases hav- enzyme function. Their P1 specificities enabled both ing little activity toward the standard synthaemtiicde proteinases to release angiotensIi1nfrom angiotensinsubstrates of tissue kallikreins wereisolated from the ogen and fromangiotensinogen I, but rK9 wasat least rat submandibular gland. This specificity has been tentatively correlated with differences in the key amino acids which define the substrate binding site in the members of the kallikrein family. NH2-terminal Sequence Analysis-Both proteinases were analyzed fortheir NH2-terminasl equence after reduction and pyridylethylation and fractionation on a C4 reverse phase chromatography column.The two peaks obtained after reduction of the Mono S-bound material corresponded to theNH2terminal sequence deduced from the nucleotide sequence of rKlk9 mRNA and to a segment starting at residue 90 (numbering based on the kallikrein rK1 sequence [5]) (Table I).

Results

Conclusion