THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267, No. 14, Issue of May 15, pp.

10045-10051,1992
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Protein Products of the Rat Kallikrein Gene Family

SUBSTRATESPECIFICITIES OF KALLIKREIN rK2 (TONIN) AND KALLIKREIN rK9*

(Received for publication, December 3, 1991)

Thierry Moreau, Michele Brillard-Bourdet, JacobBouhnikS, and Francis Gauthiert

From the Laboratoire d’Enzymologie et de Chimie des Proteines, U.R.A 1334 du Centre National de la Recherche Scientifique,
Uniuersite Frangois Rabelais, Facult6 de Midecine, Bbis, Bd Tonnelli F-37032 Tours Cedex,France and the $Unite 36 de
l’lnstitut National de la Sante et de la Recherche Medicale, 17, rue du Fer a Moulin F-75005 Paris Cedex, France

Two closely related kallikrein-like proteinases hav- enzyme function. Their P1 specificities enabled both
ing little activity toward the standard synthetic amide proteinases to release angiotensin I1 from angiotensin-
substrates of tissue kallikreins were isolated from the ogen and fromangiotensinogen I, but rK9 wasat least
rat submandibular gland. They were found to be the 100 times less active than rK2on both substrates. The
protein products of the rKlk2 (tonin) and the rKlk9 substrate specificities of rK2 andr K 9 were correlated
genes by amino acid sequence analysis (nomenclature with key amino acids definingtheir substrate binding
of the genes and proteins of the kallikrein family is site. The predicted preferentialsequence(s) around the
according to theproposal of the discussion panel from cleavage site deduced from these data may be used to
the participants of the KININ ’91 meeting held Sept. identify thebiological substrate(s)of these proteinases.
8-14,1991, in Munich, Germany). These two protein-
ases of similar structure also had very similar physi-
cochemical properties. They differed from other kal-
likrein-related proteinases in having high pHi values The protein products of the tissue kallikrein gene family
of 6.20 (rK2)and 6.86 (rK9).KallikreinrK2was have not allbeen characterized. This is mainly because of the
purified as a single peptide chain, whereas rK9 ap- large number of genes in thisfamily, particularly in the mouse
peared as a two-chain protein after reduction. Their and the rat, and the structural similarity of these genes (1-
enzymatic properties were also very similar and dif- 5). Although many of the kallikrein-related proteinases in the
fered significantly fromthose of other rat kallikrein- rat have been described, only a few have been correlated
related proteinases. Unlike the five other kallikrein- unambiguously with their corresponding gene or mRNA (6-
related proteinaseswe have purifiedso far, kallikrein 9). Tissue kallikrein (rK1)’ and tonin (rK2), encoded by the
r K 9 was not inhibited by aprotinin. r K 9 also differed genes rKlkl (formerly rGK1) and rKlk2 (formerly rGK2),
from rK2by its tissue localization. The prostate gland were thefirstto be characterized (6, 7). Four additional
contained only rK9 where it was the major kallikrein- kallikreins have recently been characterized by amino-termi-
like component. nal sequence analysis and correlated with genes in the family.
The amino acids preferentially accommodatedby the These include kallikreins rK7 and rK8, encoded by the the
proteinase 53 to 52’ subsites were identified using rKlk7 (formerly RSKG7) and rKlk8 (formerly rGK8) genes
synthetic amide and protein substrates. Unlike other (8), kallikrein rK9, formerly reported as prostatic protease
kallikrein-related proteinases, rK2 had a prevalent (9), SEV (10) or KLP-S3(11)which corresponds to theso far
chymotrypsin-like specificity, whereas rK9 had both unidentified rKlk9 gene (S3 mRNA (12)), andkallikrein rKlO
chymotrypsin-like and trypsin-like properties. Both (13), which is identical to antigen y (14) and probably to T-
rK2 and rK9 preferred aprolyl residue in position P2
of the substrate and did not accommodate bulky and kininogenase (15), for which no corresponding gene or mRNA
hydrophobic residues at that position, as did most of has so far been described.
the other kallikrein-related proteinases. This P2-pro- There is increasing interest in these proteinases because
line-directed specificity is necessary for processing the several of them have different substrate specificities, despite
precursors of several biologically active peptides. Sub- their close structural relationship, suggesting that they could
sites accommodating residues COOH-terminal to the serve different biological functions. Kallikrein rK1, for ex-
scissile bond were also important in determining the ample, is involved in processing kinin from kininogens (16),
overall substrate specificity of these proteinases. rK2 whereas rK2 can release angiotensin I1 from angiotensinogen
and r K 9 both showed a preference for hydrophobic in vitro (17). We have recently shown that kallikreins rK7,
residues in P2’. Other subsites upstream of the 53 rK8, and rKlO have substrate specificities which differ from
subsite were found to intervene in substrate binding those of rK1, particularly for residues in the P2 position
and hydrolysis. The restricted specificity of rK2 and (nomenclature of Schechter and Berger (see Ref. 18)), and
rK9 is consistent with the presence of an extended could be involved in the processing of precursors other than
substrate binding site, and hence with a processing kininogens and angiotensinogen (8, 13).
Kallikrein rK9, which has 84% amino acid identity with
* This work was supported by Grant CRE 895006 from the Institut
National de la Santk et de la Recherche MBdicale (INSERM). The rK2 (12), hasbeen reported to be a tonin-like enzyme having
costs of publication of this article were defrayed in part by the vasoconstrictive activity (10). Thesetwo proteinases therefore
payment of page charges. This article must therefore be hereby provide an excellent model for investigating structure-func-
marked “aduertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. Nomenclature of the genes and proteins of the kallikrein family
I To whom correspondence should be addressed URA CNRS 1334, is according to the proposal of the discussion panel from the partici-
Facultk de MBdecine,2bis, Bd Tonnelli., F-37032 Tours Cedex, pants of the KININ ’91 meeting held Sept. 8-14, 1991, in Munich,
France. Tel.: 33-47-36-60-45;Fax: 33-47-36-60-46. Germany.

10045
10046 Substrate Specificities of Rat Kallikreins rK2 and rK9
Characterization of rat kallikreins rK2 and rK9
M(.PP)
pHi
Reduced Nonreduced
rK2 35,000
6.20 25,000
rK9 6.85 25,000
Asterisks indicate cysteine residues which were identified as phenylthiohydantoine derivatives of pyridylethyl
cysteine.
tion relationships in this proteinase family. We have deter-
mined 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.
EXPERIMENTAL PROCEDURES~
RESULTS
Purification of Kallikreins rK2 and rK9-A rat submandib-
ular gland extract was fractionated on a DEAE-A50 column
equilibrated in 10 mM phosphate buffer, pH 6.35,O.l M NaC1.
Fractions of the two main peaks of unbound material were
assayed for their esterolytic activity usingp-tosyl-L-arginine-
methyl ester in the presence or absence of excess aprotinin.
All fractions contained esterolytic activity, but part of it,
under the firstpeak, was slightly inhibited by aprotinin (Fig.
1sin the Miniprint Section). These fractions were pooledand
fractionated on a Mono S column equilibrated in 25 mM
sodium phosphate buffer, pH 6.0. The unbound material
eluted as a single peak of aprotinin-inhibited esterolytic ac-
tivity. The bound material, which also contained esterolytic
activity, was eluted at 0.16 M NaCl and was not inhibited by
aprotinin (Fig. 2S, Miniprint Section). The two peaks were
concentrated and stored frozen until use.
NH2-terminal Sequence Analysis-Both proteinases were
analyzed fortheir NH2-terminalsequence after reduction and
pyridylethylation and fractionation on a C4 reverse phase
chromatography column.The two peaks obtained after reduc-
tion of the Mono S-bound material corresponded to theNH2-
terminal sequence deduced from the nucleotide sequence of
rKlk9 mRNA and to a segment starting at residue 90 (num-
bering based on the kallikrein rK1 sequence (5)) (Table I).
These two segments correspond to thelight and heavy chains
of the molecule, a structural feature found in the related
kallikreins rK7, rK8, rKlO (8, 13). According to the rules
proposed for kallikrein nomenclature,’ this protein, which is
identical to theprostatic protease (9), SEV (lo), and KLP-S3
(ll),will be calledkallikrein rK9.
The unbound material eluted from the reverse phase chro-
matography column as a single peak after reduction-pyridy-
lethylation, and itssequence corresponded to thatof tonin or
rK2.
Molecular Sizesand Isoelectric Points-The presence of one
chain for rK2 and two chains for rK9 was confirmed by SDS-
PAGE3 (Fig. 1).Kallikreins rK2 and rK9 appeared to be
Portions of this paper (including “Experimental Procedures,”
Figs. 1S-3S, and Table IS) are presented in miniprint a t the end of
this paper. Miniprint is easily read with the aid of a standard
magnifying glass. Full size photocopies are included in the microfilm
edition of the Journal that is available from Waverly Press.
The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate-
polyacrylamide gel electrophoresis; Abz, o-aminobenzoyb Boc, t-bu-
tyloxycarbonyl; Bz, benzoyl; EDDnp, ethylendiamine dinitrophenyl;
MCA, 7-amino-4-methylcoumarin; PECys, pyridylcysteine; PE-lyso-
zyme, pyridylethylated lysozyme; pNa, p-nitroanilide; pNPGB, p -
nitrophenyl-p’-guanidinobenzoate; Tos-Arg-OMe, p-tosyl-l-argi-
nine-methyl ester; TFA, trifluoroacetic acid.
TABLE I
NH2-terminalsequence”
IVGGYKC*EKNSQPWQVAVINEYLC*G
20,000 AYDHNNDLMLLHLSKPADITGGVK
14,000 VVGGYNC*ETNSQPWQVAVIGTTF
1 2 3 4
K9 rK9 rK2 rK2
(reduced) (redd)
Mr l o 3 X
-
94
.,
0
FIG. 1. SDS-PAGE (15%) of rat kallikrein rK2 and kalli-
krein rK9. Analytical SDS-PAGE was used to assess the purity of
rK2 and rK9 and to determine their Mrtapp). Both enzymes were
applied to 15% mini-gels (2 pg in each well) with or without prior
treatment with fl-mercaptoethanol as indicated above the gel. Molec-
ular weight markers are indicated on the left. Gel was stained with
0.25% Coomassie Brillant Blue R-250 in methanol/acetic acid/H20,
9:2:9 (v/v/v).
single chains of identical molecular mass 25 kDa prior to
reduction with 2-mercaptoethanol, whereas rK9 had two
chains of20 and 14 kDa and rK2 a single band of 35 kDa
after reduction (Table I). This unusual behavior of rK2 after
reduction has also been reported for kallikrein rK1 (8).
Kallikrein rK2 and rK9 are probably the two most basic
kallikreins present in the rat submandibular gland with pHi
values of6.20 and 6.85, respectively, as determined from
isoelectrofocusing on thin layer polyacrylamide gels using a
pH 3.5-9.5 Ampholine gradient.
Enzymatic Properties-pNPGB-titrated rK2 and rK9 were
assayed for their amidolytic and proteolytic activities using a
variety of synthetic fluorogenic substrates and native or de-
natured protein substrates. All the amide substrates tested,
corresponding to different combinations of PI, P2, and P3
residues (listed under “Experimental Procedures,” Miniprint
Section), were poorly hydrolyzed by both proteinases since
most of the kcat/Kmvalues were below 1 mM” This
suggests that either rK2 and rK9 have little amidolytic activ-
ity or that subsites other than S1 and S2, and possibly S3,
are critical for substrate binding. This possibility was inves-
tigated using protein substrates and two peptide substrates
with intramolecularly quenched fluorescence, Abz-Phe-Arg-
Ser-Arg-EDDnp and Abz-Phe-Arg-Leu-Val-Arg-EDDnp (19).
The former was significantly hydrolyzed by both proteinases
(Table 11) though at a far lower rate than that reported for
porcine or horse kallikrein (19). It was sensitive enough,
however, to measure the activity of nanomolar concentrations
of both proteinases.
Peptides and proteins, including reduced and pyridylethyl-
ated lysozyme, oxidized insulin B chain, and also substance
P, renin tetradecapeptide substrate (angiotensinogen 1-14),
and human big endothelin-1 which may be related to the
biological activity of these enzymes (both have been reported
 Substrate Specificities
Kallikreins
RatofrK2 and rK9 10047
as proteinases with vasoconstrictive activity), were then used of distribution of individual residues. Amino acids were clas-
to define the specificity of rK2 and rK9, and discriminate sified into six groups as described under “Experimental Pro-
between their activities. Bothproteinases were incubated cedures” (Miniprint Section). Results are reported in Fig. 2.
under the same experimental conditions with the same Unlike other members of the kallikrein family, rK2 and rK9
amount of each substrate. The enzyme/substrate molar ratios were able to accommodate aromatic residues in position P1.
varied between 1/15 and 1/23,000,depending on thesubstrate rK9 differed from rK2 in that it also accommodated basic
used, but were kept constant for both enzymes.Cleavage residues at that position. Both proteinases accommodated
products were separated by reverse phase chromatography on negatively charged residues in P2 but showed a preference for
a C18 column and analyzed for their NH2-terminal sequence a prolyl residue at that position. Bulky and hydrophobic
t o locate the preferential cleavage sites. As shown in Table residues, which are preferentially accommodated in S2 by
111, the two proteinases cleaved most of the substrates used kallikreins K1 and several other members of the family, did
at different sites, indicating that they have different specific- not fit the rK2 and rK9 requirements for hydrolysis. Both
ities. Their overall specificity for P3 to P2’ residues was proteinases also showed a preference for hydrophobic residues
analyzed, taking into account all rK2 and rK9 cleavage sites in P2’, which confirms the presence of an extended substrate
identified in the six peptide substrates as well as thefrequency binding site on these proteinases.
Angiotensin 11Release from Rat Angiotensinogen and from
Angiotensin I-The results obtained using the renin substrate
TABLE I1 tetradecapeptide showed that both enzymes cleaved at the
Comparison of kinetic parameters for rK2 and rK9 hydrolysis of angiotensin I1 site. The direct generation of angiotensin I1
various peptidyl-MCA derivatives and intramolecularly quenched
fluorogenic substrates from its protein precursor was further investigated by radio-
Kinetic constants were cafculated from Hanes linear plots for the
immunoassay using rat angiotensinogen as substrate. Though
peptidyl-MCA substrates and the second-order rate constant kat/K, both proteinases released angiotensin I1 but not angiotensin
for the intramolecularly quenched fluorogenic substrates was deter- I from the ratprecursor, rK9 was about 300 times less efficient
mined under first-order conditions as described under “Experimental than rK2 under the same experimental conditions. When
Procedures” (Miniprint Section). Hanes linear plotswere constructed angiotensin I was used as a substrate, rK9 was about 100
using at least six different substrate concentrations. Hydrolysis rates times less efficient than rK2 to release angiotensin I1 as
were determined in duplicate for each substrate concentration. revealed by reverse phase HPLC analysis on a C18 column.
Enzyme Km L t bJKm Inhibition by Proteinase Inhibitors-The different suscep-
p~ s” mM”.s” tibilities of rK2 and rK9 to serine proteinase inhibitors rep-
Pro-Phe-Arg-MCA 3.60 0.27
rK2 75 resent another way of discriminating between these two pro-
rK9 1.56
0.25
160 teinases. This feature was exploited to complete their purifi-
rK1 171” cation. Table IV shows the results obtained using several
Z-Phe-Arg-MCA rK2 105 0.21 2.00 representative serineproteinaseinhibitors. Only soybean
rK9 501.00
0.05 trypsin inhibitorwas as good an inhibitor of both proteinases,
rK1 57“ with apparent Kt values lower than 1 pM. Unlike all other
Boc-Leu-Ser-Thr-Arg-MCA rK23.00
0.2065 kallikrein-related proteinases reported so far (8, 20), rK2 was
rK9 48 0.06 1.25 poorly inhibited by aprotinin and rK9 was insensitive to this
inhibitor. Rat al-PI, which does not inhibit kallikrein rK1,
Boc-Ala-Gly-Pro-Arg-MCA rK2 613.60
0.22
rK91.40
0.20
146 binds both rK2 and rK9, although rather slowly. Neither rK2
nor rK9 were significantly inhibited by ovomucoid trypsin
Abz-Phe-Arg-Ser-Arg-EDDnp rK2 21.00 inhibitor, lima bean trypsin inhibitor, or chymostatin.
rK9 5.90
Abz-Phe-Arg-Leu-Val-Arg-EDDnp
rK2 0.64 DISCUSSION
rK9 0.28 In spite of their close structural similarities, with homolo-
a Ref. 8. gies of 72-89% (5),the proteinases of the rattissue kallikrein

TABLE
111
cleavage sites in various peptide and protein substrates hydrolyzed by kallikreins rK2 and rK9
Kallikrein rK2 Kallikrein rK9
Substrate
P3 P2 P1 P2’ P1’ Residueno.P2 P3 P1 PI’ P2’ Residue no.
P E lysozyme Phe Gly Arg PECys Glu 3-7 Met Lys Arg His Gly 12-16
Gly Arg Tyr Ser Leu 21-25 Asn Tyr Arg Gly Tyr 19-23
Asn Ser Arg TrpTrp 59-63 Thr Asn Arg Asn Thr 43-47
Asn Ser ArgTrpTrp 59-63
PECys Ala Lys Lys Ile 94-98
AsnArg Arg PECys112-116
Lys
Trp Ile Arg Gly PECys 123-127
Oxidized insulin
chainB AsnVal Gln His Leu Gly 2-6 Glu Arg Gly Phe 20-24
Val Glu Ala Leu Tyr 12-16
Gly Glu Arg Gly Phe 20-24
Rat angiotensinogen Pro His Phe His Leu 6-10 Pro His Phe His Leu 6-10
substrate
Renin
tetradeca-
His Pro Phe His Leu 6-10 Pro His Phe His Leu 6-10
peptide
Big-endothelin-1 (human) Val Pro Tyr Gly Leu 29-33 Val Pro 29-33
TyrLeu Gly
Substance P” Gln Gln Phe Phe5-9 Gly
Gln Phe Phe Gly Leu 6-10
a Cleavage sites of substance P by rK2 were obtained from Ref. 32.
 10048 Substrate Specificities of Rat Kallikreins rK2 and rK9

100 - HydrophW Bask (pasilivelycharged)

kreins to proteinase inhibitors have also been used to achieve
I3 Arcmatic 0 Small or noncharged their purification. In the present study, rK2 and rK9 were
80 - 0 (negatively charged)
Acidic E
i Pro, Cystine separated during their purification by their different suscep-
tibilities to aprotinin. Previously, rat kallikreins rK7 and rK8
were separated by taking advantage of the fact that rK8 is
insensitive to soybean trypsininhibitor (8). Purified rK9
occasionally showed microheterogeneity in some prepara-
tions, depending on the startingmaterial and thepurification
procedure used. Similar microheterogeneity has been reported
for kallikrein rKlO (13) and shown to be due in part to
alternative cleavage of the initial peptide chain in the region
-40 J
of the kallikrein loop (22), generating the light and heavy
P3 P2 P1’ P1 , p2’ chains of this molecule, and to avariable glycosylation of the
light chain. Such a process can be expected for kallikrein rK9,
Position which is the only other member of the family to have two
100 1 1 potential cleavage sites after Arg residues in this region, one

8ol
60 rK9
40
20
0
-20
-v
P3 p2 P1 P1‘
Position
FIG. 2. Analysis of the appearance of amino acids around
the cleaved bonds in thehydrolysis of pyridylethylated lyso-
zyme, oxidized insulin B chain, renin substrate tetradecapep-
tide, big-endothelin-1, and substance P by kallikreins rK2
and rK9. Amino acids were classified into six groups, andthe
preference for each group for P3 to P2’ positions in the substrates
was calculated as described under “Experimental Procedures.” The
cleaved bond in rat angiotensinogen, which is the same as that in
renin substrate, was not included in the calculation. Upper panel,
preference of rK2; lower panel, preference of rK9.
TABLE IV
Kinetic parametersfor the interactionof kallikreins rK2 and rK9
with some representative serine proteinase inhibitors
Apparent inhibition constants for the interaction of rK2 and rK9
with aprotinin and soybean trypsin inhibitor(SBTI) were determined
as described under “Experimental Procedures” (Miniprint Section).
The inhibition of both proteinases by a 1-proteinase inhibitor ( a 1-
PI) was characterized by the second-order rate constant for inacti-
vation k:, (see Miniprint Section for details). Resultswere the mean
of at least two independent experiments.
SBTI Aprotinin
Enzyme
K4,P) Ki,.,,,
I.” &M
rK2 0.16 76 0.4 X 10’
rK9 0.07 n.s.i.” 1.2 x lo2
a n.s.i., no significant inhibition.
family can be relatively easily separated from the same tissue
homogenate on the basis of their different electric charges.
This was first exploited by Brandtzaeg et aE. (21), who used
anion-exchange chromatography to fractionate arat subman-
dibular gland homogenate into four kallikrein-containing
pools. DEAE-A50-unbound material was used to purify four
kallikreins, including kallikreins rK2 and rK9. The other two
kallikreins, rK7 and rK8, were purified and characterized as
described previously (8). The different sensitivities of kalli-
giving rise to anNH2-terminallyblocked heavychain starting
with a Gln residue and therefore unidentifiable by sequence
analysis (13).
The amino acid sequences of rK2 and rK9 are 84% identical,
one of the highest percentages of homology between protein-
ases of the kallikrein family (12). This is most significant, as
this similarity includes the key amino acid residues thought
to be involved in the interaction with the substrate (Table
V). As a consequence, the two proteinases could be expected
to have similar functions. In accordance with this hypothesis,
Yamaguchi et al. (10) reported that kallikrein rK9, like rK2
(7, 17), has vasoconstrictive activity in uitro. However, the
P2‘ mechanism by which this activity occurs seems to be different
(10). We demonstrated by radioimmunoassay that both pro-
teinases specifically release angiotensin I1 from its precursor
and this was confirmed using asynthetic renin substrate
(angiotensinogen fragment 1-14) and angiotensin I which
were cleaved at their angiotensin I1 releasing site. However,
the total amount of angiotensin I1 released from both sub-
strates by rK9 was far lower than that released by rK2 under
the same experimental conditions, suggesting a different bio-
logical activity for each proteinase in spite of their similar
specificity.
rK2 and rK9 are similarly regulated by androgens, but their
tissue localizations differ significantly. Both are found in the
submandibular gland, but only rK9 ispresent in the rat
prostate, where it is the main kallikrein-like component. The
level of rK9 mRNA in this tissue has been reported to decrease
dramatically upon castration (9, 23), whereas it is affected
less, and to the same extent as rK2 mRNA, in the subman-
dibular gland (23). Kallikrein-related proteinases have also
been identified in thehuman anddog prostate glands (24,25).
The key amino acids of the human protease, reported as
prostate-specific antigen, differ from those in rat kallikreins
01 1-PI rK9 and rK2. In particular Asp183,which is highly conserved
kL throughout the kallikrein family, is replaced by a Ser residue
M-I.s-1
in human prostate-specific antigen (26). Other critical resi-
TABLE V
Key amino acid residues involved in the kallikrein-substrate
interaction
Positions of key amino acids that line the substrate-binding pocket
are given according to kallikrein rK1 numbering, which is used for
comparison of the amino acid sequence of the kallikrein family
proteins (Ref. 5); asterisk indicates residues of the catalytic triad.
Sources of the key residues for rK1 and rK2 are Refs. 21 and 31,
respectively.
189*
41* 183 91
96* 93 205 217
rk2 H H H D DTCA s GGAT A
rk9 H Y H D DTCT s GGAT A
rkl H D Y D DTCK s WGFN G
 Substrate Specificities of Rat Kallikreins rK2 and rK9 10049
dues such as Hisg3, GlYo5,and Ala217 in rK2 and rK9 are is generally preferred by other kallikreins (8, 13). It has been
replaced by Ser, Trp, and Gly, respectively, in human pros- suggested that a proline-directed arginyl cleavage is important
tate-specific antigen, indicating that the proteinases do not in theprocessing of peptide precursors (31). rK9, which obeys
have the same specificity and therefore serve different func- this specificity, could therefore be a good candidate for the
tions. processing of such precursors.
rK9 can be readily distinguished from the other kallikreins The differing specificities of both rK2 and rK9 from those
in the ratsubmandibular gland by its resistance to aprotinin of other members of the family agree well with the unusual
inhibition. There is, as yet, no structural feature which can structural featuresof these proteinases, and explain why they
explain this property of rK9. Both kallikreins rK2 and rK9 cannot accommodate bulky, hydrophobic residues in P2, as
are strongly inhibited by soybean trypsin inhibitor, a property do most of the otherproteinases of the family. This specificity
they share with rK7 and rKlO (8,13) but which makes them is thought to be due to thepresence of a hydrophobic sandwich
different from kallikreins rK1 andrK8, which are notsuscep- between Tyrg3and TrpZo5of the kallikreins, which traps
tible to this inhibitor. The way in which the activities of the hydrophobic P2 residues (22). Kallikreins rK2 and rK9 are
kallikrein family proteinases are regulated by physiological the only members of the family to have a glycyl residue in
proteinase inhibitors is not yet well understood; only rK1 position 205, and Tyr93 isreplaced by a histidyl residue. The
significantly modifies bloodpressure when injected in minute results reported here confirm the importance of these residues
amounts into the circulation (16). Whether or not this is for determining the size, shape and ionic character of the
related to the fact that all the kallikreins except rK1 are substrate binding pocket of kallikreins. The Gly/Ser residue
inhibited by a1-PI remains questionable, given the low rate at position 217 in most members of the kallikrein family may
at which this reaction occurs in uitro. However, an inhibitor also be important for substrate binding specificity. This resi-
of tissue kallikrein which irreversibly binds rat rK1 has been due is replaced by an Ala in rK2 and rK9, and also in rK7,
recently described (27). This kallikrein-binding protein cor- which has the same histidyl residue in position 93 (5), and
responds to thenegative acute phase reactant SPI-2 (28,29), has been shown to have a P2 specificity not restricted to
but its ability to inactivate other kallikrein-related protein- bulky, hydrophobic residues (8).
ases remains to be investigated. This approach, which allowsinvestigation of the structure-
The relationship between the structure of kallikreins and substrate specificity relationships of these two closelyrelated
their substrate specificity was investigated using a variety of proteinases, may provide a method for elucidating their bio-
peptide and protein substrates. Unlike other kallikrein pro- logical function. However, there is, to date, no evidence that
teinases, both rK2 and rK9 hydrolyze amide fluorogenic sub- the vasoconstrictor activity reported in uitro for both rK2 and
strates veryslowly, buttheyare the only two that have rK9 (10, 11, 17) is of biological relevance. With the notable
chymotrypsin-like specificity in additionto trypsin-like spec- exception of rK1, which is a physiological kinin-releasing
ificity. These two proteinases hydrolyzed the commonly used enzyme, the function of all othermembers of the ratkallikrein
kallikrein substrates Pro-Phe-Arg-MCA and Z-Phe-Arg- family also remains to be established. Identifying the phys-
MCA10-100 times slower than kallikreins rK1, rK7, rK8, iological substrates of these proteinases therefore represents
and rKlO (8, 13). Intramolecularly quenched fluorogenic pep- a considerable challenge.
tide substrates were used to study the P1’ and P2‘ specificity
of proteinases. Such substrates have been recently developed Acknowledgments-We are indebted to Dr. E. Prado (University
of Sao Paulo) for the gift of intramolecularly quenched fluorogenic
by Chagas et al. (19) to measure tissue kallikrein activity. One substrates and to Dr. T. Berg (University of Oslo) for providing
of them, Abz-Phe-Arg-Ser-Arg-EDDnp,was hydrolyzedmuch information on KLP-S3 before publication. We thank B. Cbrblis for
faster thanmethyl-coumarylamide peptide substrates by both typing the manuscript.
rK2 and rK9. However, the main substrate cleavage site, as
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266,16394-16401 201,189-198

SUPPLEhEhTARY MATERIAL TO :PROTEINPRODUCTSOF THERAT KALLIKREIN

GENE FA~TLESUBSTRATE SPECIFICITIESOF KALLIKREIN rKl qOMM AND
KALLIKREIN rK9.

Thhla?. MOREAU, MichUc BRILLARDBOURDET. Jacob BOUHNlK

and francis GAUTHlER

EXPERIMENTAL PROCEDURES

Leu-Gly-Arg-MCA. Ba-Val-Ro-Arg-MCA. N-a-B~.-Val-Oly-Arg-MCA.Bz-Phc-Vd-Arg-

MCA. Suc-AI.Z-Plo-Phs-MCA. Suc-Leu-Tyr-MCA and SuE.Lsu-Lcu-Vd.~r"CA,
chymortslin (Bachem): Boc-Ala-Gly-Pm-Alg-MCA. SBTl (Soyakan Trypsin Inhibitor).
apmfinin (Bahtinger Mannheim); human big sndothelin-1(Peptide Institu~cInc); n t al-PI
( a l - h t c i n a u Inhibitor) was punfied fmm inflammatory rat serum as d s m k d in (33); rat
sngiolsnrinogcn was purified as dcwribsd in (34): Abr-Phc-Alg-Scr-Arg-EDDnp and Ab-
Phs-Arg-Leu-V.l-Arg-EDDnp were obtained from Dr. Elins Prado (Elcola Paulista de
Medicina.SsoPaulo): all orherreagenu wreofanalyticsl grade.
Substrate Specificitiesof Rat Kallikreins rK2 and rK9 10051