Note: Descriptions are shown in the official language in which they were submitted.
CA 02301815 2000-02-23
1
NEW TISSUE-SPECIFIC CALPAINES, THEIR PRODUCTION
AND THEIR USE
The invention relates to novel tissue-specific calpains and their
preparation.
The invention furthermore relates to methods for screening for
novel calpain inhibitors and their use.
Calpains are among the intracellular, non-lysosomal enzymes of
the cysteine protease group. They are involved in Ca2*-dependent
signal transduction in eukaryotic cells, ie. they control
cellular functions in a manner dependent on the Ca2*
concentration. Calpains occur ubiquitously in animal tissues and
cells of, for example, humans, chickens, rabbits or rats.
Calpains have also been found in lower animals such as in
Drosophila melanogaster, Schistosoma or Caenorhabditis elegans.
No calpains have yet been detected in yeasts, fungi or bacteria.
To date, three main isoforms of these ubiquitous calpains are
known and differ by their calcium-dependent activatability :in
vitro. Calpain I ( _ ~ calpain) is activated by u-molar calcium
ion concentrations, whereas calpain II (= m calpain) is activated
only by millimolar concentrations of calcium ions. Both c:alpains
consist of two subunits, one large subunit of about 8U k~a and
one small subunit of about 30 kDa. Both subunits of the active
heterodimer have binding sites for calcium. The lsrge aubunit i.s
composed of the following four protein domains (= I - IV): a
protease domain (= domain II), a calcium-binding domain (= domain
IV) and two other domains (= domains I and III) whose function is
unclear. The small 30 K subunit consists of a calciun-binding
subunit (= IV') and another subunit (= V) whose function is
unclear. In addition to these two types of calpains, a 'third type
(_ ~/m 80K) which is intermediate in respect of calcium activation
has been found in chickens (Wang K.K.W. et al., TIPS, Vol. 15,
1994: 412 - 419, Suzuki, K et al., Biol. Chem. Hoppe-SAyler, Vol.
376, 1995: 523 - X29 ).
CA 02301815 2000-02-23
la
Besides these ubiquitously occurring calpains, recently two novel
tissue-specifically expressed calpains have been identified.
nCL-1 (= p94) is a muscle-specific calpain which occurs in
chickens, rats and humans and which, it is assumed, might be
active as monomer and consists only of the 80 kd subunit. Besides
nCL-1, there is a stomach-specific calpain which may occur in two
splicing variants nCL-2 and nCL-2'. nCL-2' differs from nCL-2 by
the absence of the calcium-binding region (Sorimachi, H.S, et
~
' 0050/48288 CA 02301815 2000-02-23
,.
2
al., J. Biol. Chem. Vol. 268, No. 26, 1993: 19476 - 19482,
Sorimachi, H:S: et al., FEBS Lett. 343, 1994: 1 - 5). A
calpain-homologous protein (= CalpA) which interacts with actin
and presumably plays an important part in embryonic development,
and which has two different splicing variants, has been found in
drosophila (Mol. Cell. Biol. Vol. 15, No. 2, 1995: 824 - 834). In
this case too, the shorter variant lacks the calcium-binding
site.
It is supposed that calpains play an important part in various
physiological processes. A large number of cytoskeletal,
membrane-bound or regulatory proteins such as protein-kinase C,
phospholipase C, spectrin, cytoskeletal proteins such as MAP2,
muscle proteins, neurofilaments and neuropeptides, platelet
proteins, epidermal growth factor, NMDA receptor and proteins
involved in mitosis, and other proteins, are calpain substrates
(Barrett M.J. et al., Life Sci. 48, 1991: 1659 - 69, Wang K.K. et
al., Trends in Pharmacol. Sci., 15, 1994: 412 - 419). The normal
physiological function of the calpains is, however, still not
clearly understood. They are-.involved in a large number of
physiological processes such as apoptosis, cell division and
differentiation or in embryonic development.
Elevated calpain levels have been measured in various
pathophysiological processes and disorders, for example in:
ischemias of the heart (eg. myocardial infarct), of the kidney or
of the central nervous system (eg. stroke), inflammations,
muscular dystrophies, cataracts in the eyes (gray cataract),
injuries to the central nervous system (eg. trauma), Alzheimer's
disease, HIV-induced neuropathy, Parkinson's and Huntington's
diseases etc. (see Wang K.K. above). It is assumed that there is
a connection between these disorders and an elevated and
sustained intracellular calcium level. This results in
calcium-dependent processes being overactivated and no longer
subject to physiological control. Correspondingly, overactivation
of calpains may also initiate pathophysiological processes.
This is why it has been postulated that inhibitors of calpain
enzymes may be useful for treating these disorders. Various
investigations have confirmed this. Thus, Seung-Chyul Hong et al.
(Stroke 1994, 25 (3), 663 - 669) and Bartus R.T. et al.
(Neurological Res. 1995, 17, 249 - 258) have shown that calpain
inhibitors have a neuroprotective effect in acute
neurodegenerative disturbances as occur after stroke. Likewise,
following experimental brain injuries, calpain inhibitors
improved the recovery from the memory deficits and neuromotor
disturbances occurring (Saatman K.E. et al., Proc. Natl. Aced.
~
' 0050/48288 CA 02301815 2000-02-23
r~
3
Sci. USA, 93, 1996: 3428 - 3433). Edelstein C.L. et al. (Proc.
Natl. Acad. Sci. USA, 92, 1995, 7662 - 7666) found that calpain
inhibitors have a protective effect on kidneys damaged by
hypoxia. Yoshida K.I. et al. (Jap. Circ. J. 59 (1), 1995, 40 -
48) were able to show that calpain inhibitors have beneficial
effects after cardiac damage caused by ischemia or reperfusion.
Since calpain inhibitors inhibit the release of (3-AP4 protein, a
potential use for therapy of Alzheimer's disease has been
proposed (Higaki J. et al., Neuron, 14, 1995: 651 - 659). The
release of interleukin-la is likewise inhibited by calpain
inhibitors (Watanabe N. et al., Cytokine, 6 (6), 1994: 597 -
601). It has furthermore been found that calpair. inhibitors have
cytotoxic effects on tumor cells (Shiba E, et al., 20th Meeting
Int. Ass. Breast Cancer Res., Sendai Jp, 1994, 25. - 28. Sept.,
Int. J. Onco. 5 (Suppl.), 1994, 381). Calpain also plays an
important part in restenosis and in arthritis, and calpain
inhibitors may have a beneficial effect on the pathology (March
K:L: et al. Circ. Res. 72, 1993: 413 - 423, Suzuki K. et al.,
Biochem J., 285, 1992: 857 - 8f2).
Further possible uses of calpain inhibitors are to be found in
Wang K.K (Trends in Pharmacol. Sci., 15, 1994: 412 - 419.).
Thz most potent and selective calpain inhibitor is the naturally
occurring intracellular proteir. calpastatin. It inhibits both -.
calpain I and calpain II, but not other cysteine and thiol
proteases such as cathepsin B, L or papain. However, calpastatin
has the disadvantage that it is unsuitable for possible therapies
because of its size, consisting of about 700 amino acids, and
inability to cross the cell membrane. Besides low molecular
weight calpain inhibitory peptides, a number of non-peptide
inhibitors have been identified. The disadvantages of these
inhibitors are that they are unstable, are rapidly metabolized
and some of them are toxic. Many calpain inhibitors additionally
have insufficient selectivity, ie. they inhibit not only calpain I
and II but also other cysteine proteases such as papain,
chymotrypsin, elastase or cathepsin B and L.
Thus there continues to be a need for selective and highly
effective calpain inhibitors. Highly specific test systems are
needed for screening for these selective and very effective
calpain inhibitors to allow selective inhibitors to be
identified. The screening tests are usually carried out with the
ubiquitously occurring calpain I and calpain II.
~
' 0050/48288 CA 02301815 2000-02-23
,,
4
To find selective inhibitors, it is necessary and desirable to
provide further calpains which are expressed as
tissue-specifically as possible for the testing, so that the
inhibitors can be tested for their selectivity between the
individual calpains.
In addition, further novel calpains are sought because they are
proteins which very probably are expressed differently in various
pathologies and disorders and play an important part in these
disorders.
It is an object of the present invention to provide means for
distinguishing and identifying calpain inhibitors which make it
possible to identify calpain inhibitors which, on the one hand,
have an inhibitory effect on only one calpain and/or, on the
other hand, have an inhibitory effect on a plurality of calpains,
and to provide these as therapeutic target.
47e have found that this object is achieved by a novel
tissue-specific calpain gene having the sequence SEQ ID N0: 1 or
SEQ ID N0. 3 and the name CAPN6, its allelic variants, analogs or
derivatives which, at the derived amino acid level, have a
homology of from 60 to 100 %, where the calpain genes,.their
allelic variants, analogs or derivatives contain the following
aequances:
(a) Leu-Gly-Asn-Lys-Ala,
where this sequence differs from the corresponding sequence
in human calpain I in that the amino acid cysteine in human
calpain I, which occupies position 115 in calpain I, is
altered to lysine which is position 81 in sequences SEQ ID
No. 1 and SEQ ID No. 3;
(b) Ala-X-Ser-Cys-Leu-Ala,
where, compared with the corresponding sequence in human
calpain I, the amino acids alanine and threonine in
positions 122 and 125 are altered to serine and alanine in
positions 88 and 91 in sequences SEQ ID No. 1 and SEQ ID No.
3;
(c) Gly-Tyr-Thr-(His oder Tyr)-Thr-X-Thr,
where, compared with the corresponding sequence in human
calpain I, the amino acids histidine, alanine and serine in
positions 272, 273 and 275 are altered to tyrosine, threonine
and threonine in positions 252, 253 and 255 in sequences SEQ
ID No. 1 and SEQ ID No. 3 and in SEQ ID No. 3 additionally
,.
0050/48288 CA 02301815 2000-02-23
,.
the tyrosine residue in position 274 in calpain I is altered
to histidine in position 254 in SEQ ID No. 3;
(d) Arg-X-Arg-Asn-Pro-Leu-Gly
5 where this sequence differs from the corresponding sequence
in human calpain I in that the amino acid tryptophan in human
calpain I, which occupies position 298 in calpain I, is
altered to leucine which is in position 286 in sequences SEQ
ID No. 1 and SEQ ID No. 3, and
X in said sequences is any natural amino acid.
The invention also relates to a method for identifying calpain
inhibitors, where a calpain, its allelic variants or analogs
encoded by a sequence as claimed in claim 1 is isolated from
tissues or cells, and the inhibition of the cleavage of a
substrate of the enzyme CAPN6 and, in at least one other test,
the inhibition of the cleavage of a substrate of the enzymes
calpain I and/or II by. test substances are measured, and the test
substances which inhibit the enzyme CAPN6 and at least one other
calpain are selected.
The invention furthermore relates to a method for identifying
.calpain inhibitors, wherein the inhibition of the cleavage of a
substrate of the enzyme CAPN6 or of the calpains I and/or II by
test substances in cellular systems is determined, and the test
substances which cross the cell membrane and inhibit the
intracellular activity of the enzyme CAPN6 and/or of calpains I
and/or II, which do not inhibit the enzyme CAPN6 but the enzymes
calpain I and/or II, or which inhibit the enzyme CAPN6 but not
the enzymes calpain I and/or II are selected. Substances which
show an in vitro activity toward the calpains without their
ability to enter the cell having been tested are also
advantageously selected. If a subsequent assay shows that these
substances are unable, or only poorly able, to enter the cell,
their cell permeability can be improved by derivatization.
Human ~t and m calpain protein gene sequences were used for a
search for homology in the EST databank of the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov) using
the BLAST method program. A sequence called EST AA050030 which
has a sequence typical of calpains was found. It was possible
with the aid of this sequence to prepare a mouse clone which
codes for a gene whose novel gene product was, as novel calpain,
called CAPN6 (= nCL-4). The nucleic acid sequence of the clone
nCL-4 is to be found in SEQ ID N0: 1. The derived amino acid
sequence of the calpain CAPN6 is to be found in SEQ ID NO: 2. The
~
' 0050/48288 CA o23oisis 2000-02-23
6
amino acid sequence, which was deduced taking account of the
presence of an intron, shows a typical calpain signature,
although assignment to the known calpain subfamilies of ~.
calpain, m calpain, nCL-1 or nCL-2 is not possible because of the
low homology. The calpain CAPN6 is a novel, previously unknown,
calpain. Further investigations using this sequence from mice in
the EST databank provided in addition 4 human part-sequences
(AA169715, C17331, C16980 and T39424) having a homology with the
mouse CAPN6 sequence. These part-sequences could be assembled
into a continuous sequence coding for a protein with a length of
374 amino acids. This sequence lacks both the typical start codon
for methionine and the stop codon. This sequence is therefore
probably only part of the sequence of the human ortholog to the
cloned mouse sequence. The homology of the two sequences of the
374 amino acids is 94.9% (Figure 1).
The protein sequence derived from the gene sequence SEQ ID N0: 1
shows 30% homology, which is the greatest homology, with the
. known Caenorhabditis elegans gene tra-3 over the entire amino
acid sequence. The homology with the other known calpains is from
20.9% (rat nCL-2) to 25.4% (mouse m calpain). In a sequence
comparison by the Lipman-PearsQn method (Ktupl 2, Gap Penalty 4,
Gap Length Penalty 12) between CAPN6 and human CAPN~ (_
CAN1 HUMAN, Aoki et al., FEBS Lett. 205, 1986: 313 - 317), human
CAPN2 (= CAN2-HUMAN, Aoki et al., Biochemistry 27, 1988: 3122 -
8128), rat CAPN2 (= CAN2 RAT, Deluca et al., Biochim. Biophys.
Acta 1216, 1993: S1 - 93), human CAPN3 (= CAN3 HUMAN, Richard et
al., Cell 81, [lacuna] 27 - 40) rat CAPN3 (= CANS RAT, Sorimach'i
et al., J. Biol. Chem. 264, 1989: 20106 - 20111) and Drosophila
calpain (= DMCLPNOCM_1) over part-sequences of 230 - 520 amino
acids, slightly greater homologies of from 32.8 to 39.5% were
found, but these overall are less than the homologies with tra-3
(Figure 1, alignment, Clustal method with PAM25 residue weight
tabl.). Besides tra-3, CAPN6 shows pronounced homology with the
recently described CAPN5 calpain (44.2% homology, File No.
P 19 718 248. 8 ) .
Sequence comparisons between the mouse and human CAPN6 sequence
in a wide variety of databanks revealed homologies with CalpA,
Tra-3 and human sequences called C16980, T39424, AA16971, 893331
and 617331, about whose functions no information was provided.
The sequence comparisons were carried out using the gene bank EST
and databanks at the National Center for Biotechnology
Information (http: Hwww.ncbi.nlm.nih.yor [sic]) and the Wash-U
databank (Homo Sapiens). Also found in the databanks was a mouse
EST sequence with the name AA050030 and designated as calpain. No
further information could be obtained from the databanks. The
~
' 0050/48288 ca o23oisis 2000-02-23
7
complete gene sequences of AA16971T, 893331, C17331 and AA050030
are unknown.
The two calpains (CAPNS and CAPN6) have properties in common
which distinguish them from other calpains. CAPN5 and CAPN6 have,
by comparison with the other calpains, not only a truncated
domain I but also a modified C-terminal end which has no extensive
homology with domain IV of the other calpains. The consensus
sequence of the Ca2+- binding site of the calpains (called the EF
hand) is located in the region of domain IV. This Ca2+- binding
site is absent in the case of CAPNS and CAPN6, which means that
possibly no Ca2+ is bound to domain IV, and the proteins are
activated in another way. They are thus the only vertebrate
calpains lacking the calmodulin-like domain IV.
The derived CAPN6 amino acid sequence has another peculiarity in
the form of a modified catalytic center. Only the Asn residue in
position 284 is conserved. The cysteine residue in position 81 and
the histidine residue in position 252 have been respectively
replaced by lysine and tyrosine in the mouse CAPN6 sequence. In
addition, further replaced amino acids have been identified in
the region of the catalytic center on comparison of the CAPN6
sequences SEQ ID No. 1 and SEQ ID No. 3 with human calpain I
(= CAPNl, Aoki et al., FEBS Lett. 205, 1986: 313 -317). Thus, the
amino acids alanine and threonine in positions 122 and 125 in
CAPN1 are altered to serine and alanine in positions 88 and 91 in
CAPN6. There are likewise alterations of amino acids histidine,
alanine, serine and tryptophan in positions 272, 273, 275 and 298
in CAPN1 to tyrosine, threonine, threonine and leucine in
positions 252, 253, 255 and 286 in CAPN6. Assignment of the amino
acids to the various positions in the proteins CAPN1 and CAPN6
takes place by comparison of the sequences and assignment of the
conserved regions of the proteins relative to one another for
maximum agreement between the proteins at the amino acid level.
It is thus possible, for example, for the same sequences to be
located at very different sites or positions in different
proteins in a protein family.
The human CAPN6 sequence has, in addition to the replacement of
the cysteine residue, a replacement of the tyrosine in
position 254 by histidine (Table 1). A possible explanation might
be, for example, that CAPN6 has a substrate specificity which is
different from the other calpains, or the active center is not
critical for the CAPN6 function, that it is an inhibitor of other
calpains, or that CAPN6 is a pseudogene. This last possibility
appears to be improbable because of the large number of conserved
amino acids. It is probable that the cysteine residue in
'' 0050/48288 ca o23oisis 2000-02-23
8
position 89 can take over the function of the missing cysteine
residue in position 81 in the catalyzed reaction. Even if CAPN6
had no protease activity, which is very unlikely, it might be
involved in regulatory processes, possibly by competing with
other calpains for binding sites on cofactors or substrates.
Table 1: CAPN6 genomic sequences
Amino acid No. 81* ~8~9 254* 284* 2861
Mouse CAPN6 (129 ES cells DNA) K C Y N L
Human CAPN6 (HeLa cells DNA) K C H N L
Other calpains C S/C Y N W
* Amino acids of the active center (Arthur et al., FEBS Lett.
368, 1995: 397 - 400)
1 low activity without leucine in m calpain (Arthur et al.,
FEBS Lett. 368, 1995: 397 - 400)
Tra-3 is involved in sex determination in Caenorhabditis elegans.
A cascade of several genes and their gene products involving
tra-3 decides on whether caenorhabditis males or hermaphrodites
develop (Kuwabara P.E. et al., TIG, Vol. 8, No.S, 1992: 164 -
168). Tra-3 appears to be involved in spermatogenesis. The
conservation of amino acids in the various domains between mouse
CAPN6 and tra3 [sic] is 39.2%, 42.0%, 30.9% and 22% respectively
for domains I, II, III and T.
Starting from cDNA derived from (Ia) 17 mouse embryo mRNA it was
possible with the aid of a modified RACE (= rapid amplification
of cDNA ends) method of Frohman et al. (Proc. Natl. Acad. Sci.
USA 85, 1988, 8998 - 9002) and Edwards et al. (Nucl. Acids Res.
19, 1991, 5227 - 5232) and using the abovementioned primers (Cal6
and Cal9) and sequences derived from EST AA050030 to clone the
complete sequence of the cloned CAPN6. SEQ ID No. 1 codes for a
protein with 641 amino acids and a molecular weight of 74.6 kDa.
The methionine start codon is preceded by a typical sequence for
the start of translation. The correctness of the sequence was
confirmed by sequencing several cDNA clones having said sequence.
Figure 2 shows the homologies between mouse CAPN5 (= nCL-3),
human CAPNS (= nCL-3), mouse CAPN6 (= nCL-4) and human CAPN6
(= nCL-4). Figure 2 also shows the sequences of caenorhabditis
tra-3, human p94, mouse m calpain, human ~t calpain and rat nCL-2.
Amino acids which agree between the various calpains and CAPN6
are indicated by black boxes. Dashes indicate gaps introduced to
achieve maximum agreement of the sequences. For clarity, two
sequences have been deleted from the human p94 sequence. These
~
' 0050/48288 ca o23oisis 2000-02-23
9
regions have been indicated by =. The conserved amino acids in
the catalytic center have been marked with arrows. The amino acid
sequences corresponding to CAL6 and CAL9 have been underlined.
The domain designations have been indicated above the relevant
sequence segments.
Figure 3 shows the phylogenetic pedigree of the various calpains.
The phylogenetic analyses to construct this pedigree were carried
out using the nearest neighbor method (Saitou et al. Mol. Biol.
Evol. 4, 1987, 406 - 425) excluding the gaps. It was possible
with the aid of these phylogenetic analyses to divide the
vertebrate calpains into six different groups (Figure 3,
right-hand side). The invertebrate calpains can be assigned as
nearest neighbors to the CAPNS-(= nCL-3-) and CAPN6-(= nCL-4-)
group and form their own group. The CAPN5 and CAPN6 genes thus
each form their own group of calpains which have a greater
similarity to invertebrate calpains than to vertebrate calpains.
The length of the horizontal lines is proportional to the
phylogenetic distance between the various calpains. The length of
the vertical lines has no significance. The sequences used to
construct the phylogenetic pedigree have the following SWISSPROT
and EMBL numbers (accession numbers): human m (P17655), ~t
(P07384), p94 (P20807); rat m (Q07009), nCL-2 (D14480), p94
(P16259); mouse p94 (X92523); chicken m (D38026), ~u (D38027), ~,/m
(p00789), p94 (D38028); nematode tra-3 (U12921); Drosophila Calp
A (Q11002) and Dm (X78555), Schistosoma (P27730). The human nCL-2
part-sequence corresponds to translation of the EST clone
AA025030 (Hellier et al., 1995, The Wash U-Merck EST project).
The derived amino acid sequence of the EST clone AA026030
displays a greater than usual homology with the rat nCL-2
sequence according to this analysis. Since only a part-sequence
was used for this analysis, the inaccuracy of the phylogenetic
pedigree obtained for nCL-2 is greater. The nematode CPL1
sequence is the corrected version used by Barnes and Hodjkin
(EMBOJ., 1996, 15:4477-4484).
The first 7 exons were used as they can be obtained from the EMBL
data bank (Accession No. L25598), while the last 5 exons were
obtained by connecting the nucleotides 8028-8133, 8182-8239,
8729-8818, 8865-8983 and 9087 up to the end. The derived
N-terminal sequence is unusual for calpains on account of its
length and its many glycine residues.
The novel tissue-specific calpain CAPN6 is expressed only in
placental tissue (Figure 4). The human placenta is an organ which
shows fast growth and rapid differentiation. It is therefore also
~
' 0050/48288 ca o23oisis 2000-02-23
referred to as "premalignant tissue" because it is a highly
invasive tissue similar to that of malignant tumors.
Proteases play a central part in the development and
5 differentiation of cells and thus also in the placenta. The
placenta is rich in proteases and protease inhibitors which, in a
balanced equilibrium, make it possible for the placenta to
develop. CAPN6 appears to play an important part in this as
protease and/or is possibly involved in the regulation of other
10 calpain cysteine proteases.
It is possibly involved in pathological processes of the placenta
such as gestosis (= preeclampsia) which occurs in 5 to 7~ of all
pregnancies. Preeclampsia (= high blood pressure induced by
pregnancy) is one of the commonest complications of pregnancy and
is characterized by hypertension and proteinuria, often combined
with excessive edemas and, in some circumstances, with
convulsions. Preeclampsia during pregnancy is an important cause
of death of mother and child, of premature births or, in milder
cases, of embryonic malnutrition or growth disturbances. It is a
multifactorial event in which there is involvement of, for
example, genetic factors (recessive or dominant genes), the
mother's immune tolerance, vasodilator/vasoconstrictor imbalances
and, presumably, also CAPN6.
Women suffering from preeclampsia have altered vasopressinase and
angiotensinase levels which are possibly influenced in a
regulatory manner by CAPN6.
Another possible function of CAPN6 might be regulation of the
breakdown of somatostatin, glucagon and growth hormone in the
placenta and thus influence growth of the fetus. The breakdown of
maternal serum proteins which likewise stimulate growth of the
fetus might also be influenced by CAPN6.
It is possible that CAPN6 is involved in complex events in the
placental mucosa during embryogenesis and thus controls the
apoptosis induced by TNFa and IFNy, for example of the
trophoblasts by regulating other calpains. CAPN6 thus appears to
be involved in embryonic development.
In order to identify selective calpain inhibitors there is a need
for methods which are as specific as possible for identifying the
inhibitors. It is important in this connection that the selected
inhibitors inhibit only the required calpain(s) but not other
cysteine proteases and thus intervene in physiological processes.
' 0050/48288 ca o23oisis 2000-02-23
11
The test substances to be investigated for their inhibitory
activity may be, for example, chemical substances, microbial or
plant extracts. Besides the test for their inhibitory activity on
CAPN6, calpain I and/or II, they are normally also tested for
their activity toward cathepsin B or other thiol proteases.
Ideally, good inhibitors should show little or no activity toward
cathepsin B, L, elastase, papain, chymotrypsin or other cysteine
proteases but show good activity toward calpains I and II. -
It is possible with the novel tissue-specific calpain CAPN6 to
use the method according to the invention to identify inhibitors
which are able to discriminate in their inhibitory action between
the various calpains comprising calpain I, II, nCL-1, nCL-2 and/or
nCL-4.
The various inhibitor 'tests were carried out as follows for this
purpose:
Cathepsin B test
Cathepsin B inhibition was determined by a method similar to that
of S. Hasnain et al., ~. B.iol. Chem. 268, 1993, 235 - 240.
2 ~tl of an inhibitor solution prepared from the chemical
substance to be tested, a microbial or plant extract and DMSO
(final concentration: 100 ~M to 0.01 ~M) are added to 88 ~1 of
cathepsin B (cathepsin B from human liver supplied by Calbiochem,
diluted to 5 units in 500 ~,M buffer). This mixture is preincubated
at room temperature (= 25°C) for 60 minutes and then the reaction
is started by adding 10 ~1 of 10 mM Z-Arg-Ar.g-pNA (in buffer with
10% DMSO). The reaction is followed in 3 microtiter plate reader
at 405 nm for 30 mintes. The IC5o values are then determined from
the maximum slopes.
Calpain I and II test
The activity of the calpain inhibitors was investigated in a
colorimetric test with Hammarsten casein (Merck, Darmstadt)
as substrate. The test was carried out in microtiter plates in
accordance with the publication by Buroker-Kilgore and Wang in
Anal. Biochem. 208, 1993, 387 - 392. The enzymes used were
calpain I (0.04 U/test) from erythrocytes and calpain II
(0.2 U/test) from kidney, both from pigs, supplied by Calbiochem.
The substances to be tested were incubated with the enzyme at
room temperature for 60 minutes, the concentration of the solvent
DMSO not exceeding 1%. After addition of the Bio-Rad color
reagent, the optical density was measured at 595 nm in an SLT Easy
0050/48288 ca o23oisis 2000-02-23
12
Reader EAR 400. The 50% activity of the enzyme is evident from the
optical densities determined at the maximum activity of the
enzyme without inhibitors and the activity of the enzyme without
addition of calcium.
The activity of calpain inhibitors can furthermore be determined
using the substrate Suc-Leu-Tyr-AMC. This fluorimetric method is
described by Zhaozhao Li et al., J. Med. Chem. 36, 1993,
3472-3480.
Since calpains are intracellular cysteine proteases, it is
necessary for calpain inhibitors to cross the cell membrane in
order to prevent breakdown of intracellular proteins by calpain.
Some known calpain inhibitors such as E 64 and leupeptin overcome
the cell membranes only poorly and, accordingly, show only a poor
effect on cells although they are good calpain inhibitors. It is
therefore advantageous to carry out an additional test of the
ability of potential calpain inhibitors to cross membranes, such
as the human platelet test.
Platelet test to determine the cellular activity of calpain
inhibitors
The calpain-mediated degradation of proteins in platelets was
carried out as described by Zhaozhao Li et al., J. Med. Chem.,
36, 1993, 3472- 3480. Human platelets were isolated from fresh
sodium citrate blood from donors and adjusted to 10~ cells/ml in
buffer (5 mM hepes, 140 mM NaCl and 1 mg/ml BSA, pH 7.3).
Platelets (0.1 ml) were preincubated in 1 ~.1 of various
concentrations of potential inhibitors (dissolved in DMSO) for
5 minutes. Then calcium ionophor A 23187 (1 ~,M in the test} and
calcium (5 mM in the test) were added and a further incubation
took place at 37°C for 5 minutes. After a centrifugation step, the
platelets were taken up in SDS-page sample buffer and boiled at
95°C for 5 minutes, and the proteins were fractionated in an 8%
gel. Breakdown of the two proteins actin-binding protein (= ABP)
and talin was followed by quantitative densitometry. After
addition of calcium and ionophore, these proteins disappeared,
and new bands with a molecular weight of less than 200 Kd
appeared. The half-maximum enzyme activity with or, as control,
without inhibitor is determined therefrom.
Likewise suitable for testing the ability to cross membranes are
parts of. tissues such as brain sections or cell cultures.
~
' 0050/48288 ca o23oisis 2000-02-23
13
The test for inhibition of CAPN6 is carried out in cells which
express this protein and allow the latter to be detected using a
specific antibody. If cells are stimulated with, for example,
calcium and the appropriate ionophor, this leads to activation of
CAPN6. Takaomi Saido described in J. Biochem. 11, 1992, 81-86 the
autolytic transition of ~. calpain after activation and detection
using antibodies. Appropriate antibodies for detecting CAPN6 are
generated. Calpain inhibitors prevent the autolytic transition
and corresponding quantification is possible using antibodies.
Besides the described in vitro tests and the cellular platelet
test, all other calpain tests known to the skilled worker are
suitable, such as the test of inhibition of glutamate-induced
cell death in cortical neurones (Maulucci-Gedde M.A. et al.,
J. Neurosci. 7, 1987: 357 - 368), the calcium-mediated cell death
in NT2 cells (Squier M.K.T. et al., J. Cell. Physiol., 159, 1994:
229 - 237, Patel T. et al., Faseb Journal 590, 1996: 587 - 597),
or analysis of tissue samples for breakdown products of proteins
such as spectrin, MAP2 or tau (Ami Arai et al., Brain Research,
1991, 555, 276 - 280, James Brorson et al., Stroke, 1995, 26,
1259 - 1267).
For in vitro tests of CAPN6, the calpain or its animal or human
homolog is purified from tissues or cells in which the enzyme is
normally or artificially (eg. by recombinant expression)
expressed such as, advantageously, the placenta or from ceps or
microorganisms which contain at least one gene copy and/or a
vector having at least one copy of the CAPN6 gene, its allelic
variants or analogs, and used as crude extract or as pure enzyme.
For the methods according to the invention, the various calpain
inhibitor tests are advantageously carried out in combination
with the test for inhibition of CAPN6 enzyme activity by
potential inhibitors. This entails selection of inhibitors which
inhibit either only the enzyme CAPN6 and not the other calpains
or, conversely, only the other calpains and not the enzyme CAPN6
or the enzyme CAPN6 and at least one other calpain. Inhibitors of
CAPN6 can advantageously be used in the event of gestosis.
The various inhibitor tests are moreover carried out in such a
way that, in addition to the test for the inhibiting effect of
the test substance on CAPN6, calpain I and/or II, as a control
the tests [sic] is carried out without the test substance. This
test arrangement makes it easy to detect the inhibitory effects
of the test substances.
0050/48288 CA 02301815 2000-02-23
14
Another method according to the invention uses CAPN6 or its
allelic variants, analogs or synthetic derivatives advantageously
for protection from the enzymatic action of other calpains.
Another method according to the invention uses the enzyme CAPN6
for screening for novel calpain inhibitors, these inhibitors
advantageously being able to inhibit all calpains in general or
individual calpains such as calpain I, II, nCL-1, nCL-2 or CAPN6.
The various test substances can moreover be tested singly or in
parallel in test systems. The test substances are advantageously
screened for their inhibitory action in parallel automated test
systems.
In general, all substances are suitable for the inhibitor tests.
Thus, the substances are derived, for example, from classical
chemical synthesis, from combinatorial chemistry, or from
microbial, animal or plant extracts. blicrobial extracts mean, for
example, fermentation broths, disrupted cells of microorganisms
or substances after biotransformation. Cell fractions are also
ZO suitable for the tests.
Suitable for cloning the CAPN6 gene or its animal homologs or its
human homologs, its allelic variants or analogs are all
prokaryotic or eukaryotic expression systems suitable for
isolating an enzymatically active gene product. Preferred
expression systems are those which allow expression or the CAPN6
gene sequences in bacterial, fungal or animal cells, very
particularly preferably in insect cells. Enzymatically active
gene product means CAPN6 proteins which afford, directly after
isolation from the expression organism, for example from a
prokaryotic or eukaryotic cell, or after renaturation, an active
protein which is able to cleave at least one known calpain
substrate like those mentioned above, or itself by autocatalysis.
Tests suitable for determining the enzymatic activity of calpain
are all those known to the skilled worker, such as in vitro tests
like the tests described above for calpain I and II or cellular
tests such as the platelet test. A possibility which can be used
for detection are tests based on the basis of a colorimetric
assay (Buroker-Kilgore M. et al., Anal. Biochem. 208, 1993: 387 -
392) or of a fluorescence assay.
In addition, the enzymatically active gene product of CAPN6 also
means all part-sequences which contain the catalytic center of
the CAPN6 gene and/or other sequences of the CAPN6 gene and/or
005048288 CA 02301815 2000-02-23
other calpain gene sequences and/or other sequences and show
enzymatic activity.
By host organisms are meant all prokaryotic or eukaryotic
5 organisms suitable as host organisms, for example bacteria such
as Escherichia coli, Bacillus subtilis, Streptomyces lividans,
Streptococcus carnosus, yeasts such as Saccharomyces cerevisiae,
Schizosaccharomyces pombe, fungi such as Aspergillus niger,
insect cells such as Spodoptera frugiperda, trichoplusia cells or
10 all other insect cells suitable for viral expression, or animal
cells such as CV1, COS, C127, 3T3 or CHO or human cells.
By expression systems are meant the combination of the expression
organisms mentioned above by way of example, and of the vectors
15 suitable far the organisms, such as plasmids, viruses or phages
such as the T7 RNA polymerase/promoter system or vectors having
regulatory sequences for phage ~.
The term expression systems preferably means the combination of
Escherichia coli and its plasmids and phages or the baculovirus
system and the corresponding insect cells such as Spodoptera
frugiperda.
In addition, further 3' and/or 5' terminal regulatory~sequences
are suitable for the advantageous expression according to the
invention of the CAPN6 gene.
These regulatory sequences are intended to make specific
expression of the CAPN6 gene possible. This may mean, for
example, depending on the host organism, that the gene is
expressed or overexpressed only after induction, or that it .is
immediately expressed and/or overexpressed.
The regulatory sequences and factors may moreover preferably have
a beneficial effect on, and thus increase, CAPN6 gene expression.
Thus, the regulatory elements can be enhanced, advantageously at
the level of transcription, by using strong transcription signals
such as promoters and/or enhancers. However, it is also possible
besides this to enhance translation by, for example, improving
the stability of the mRNA.
Enhancers mean, for example, DNA sequences which bring about
increased CAPN6 gene expression via an improved interaction
between RNA polymerase and DNA.
, 005048288 CA 02301815 2000-02-23
16
One or more DNA sequences may be present in front of and/or
behind the CAPN6 gene with or without promoter in front, and with
or without regulatory gene, so that the gene is present in a gene
structure.
Expression of the CAPN6 gene can moreover be increased by
increasing the copy number of the CAPN6 gene. The copy number of
the CAPN6 gene is increased, for example, by amplification in a
CHO expression vector. Suitable vectors are also vectors of the
pED series - dicistronic vectors - which also contain the
amplifiable marker gene of dihydrofolate reductase. Details can
be found in Current Protocols in Molecular Biology Vol. 2, 1994.
An increase in the CAPN6 enzyme activity by comparison with the
starting enzyme can be achieved, for example, by modifying the
CAPN6 gene or its animal homologs by classical mutagenesis such
as UV irradiation or treatment with chemical mutagents and/or by
specific mutagenesis such as site-directed mutagenesis,
deletion(s), insertions) and/or substitution(s). The enzyme
activity can be increased, for example, by modifying the
catalytic center so that there is faster conversion of the
substrate to be cleaved. An increased enzyme activity can also be
achieved, besides the gene amplification described, by
eliminating factors which repress enzyme biosynthesis and/or. by
synthesizing active in place of inactive CAPN6 proteins. It i~
possible in this way to provide increased amounts of enz~,rmes for
the in vitro tests.
CAPN6 or its animal homologs or its human homolog can
advantageously be cloned starting from genomic DNA or cDNA using,
for example, the PCR technique (see Molekular Cloning, Sambrook,
Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press,
Second Edition 1989, Chapter 14, 1 - 35, ISBN 0-87969-309-6 and
Saiki et al., Science, 239, 1988, 487 et seq.), and CAPN6 can
preferably be cloned using genomic DNA, and particularly
preferably using genomic DNA from mouse cells or human cells.
Suitable as host organism for the cloning, for example, are all
Escherichia coli strains, preferably the Escherichia coli strain
DH10B. Suitable vectors for the cloning are all vectors suitable
for expression in Escherichia coli (see Molecular Cloning,
Sambrok [sic], Fritsch and Maniatis, Cold Spring Harbor,
Laboratory Press, Second Edition 1989, ISBN 0-87969-309-6).
Particularly suitable examples are vectors derived from pBR or
pUC, or shuttle vectors, and pBluescript is very particularly
suitable.
' 0050/48288 CA 02301815 2000-02-23
17
Obtainable after isolation and sequencing are CAPN6 genes having
nucleotide sequences which code for the amino acid sequence
indicated in SEQ ID NO: 2, or its allelic variants. By allelic
variants are meant CAPN6 variants which have from 60 to 100%
homology at the amino acid level, preferably 70 to 100%, very
particularly preferably 80 to 100%. Allelic variants comprise, in
particular, functional variants obtainable from the sequence
depicted in SEQ ID NO: 1 or SEQ ID NO: 3 by deletion, insertion or
substitution of nucleotides, but where the CAPN6 activity is
retained and the sequences (a) Leu-Gly-Asn-Lys-Ala, (b)
Ala-X-Ser-Cys-Leu-Ala, (c) Gly-Tyr-Thr-(His or Tyr)-Thr-X-Thr and
(d) Arg-X-Arg-Asn-Pro-Leu-Gly are present as also in the CAPN6
genes, analogs or derivatives.
By analogs of CAPN6 are meant, for example, its animal homologs,
truncated sequences, single-stranded DNA or RNA of the coding and
noncoding DNA sequence, in particular antisense RNA.
Examples of CAPN6 derivatives are those derivatives which can be
cleaved enzymatically only with difficulty, if at all, such as
the nucleic acid phosphonates or phosphothioates in which the
phosphate group ~f the nucleic acids has been replaced by a
phosphonate or thioate group.
The promoter which is present in front of the stated nucleotide
sequence can also be modified by one or more nucleotide
exchanges, by insertions) and/or deletion(s), but without
impairing the functionality or activity of the promoter. The
promoter may furthermore have its activity increased by
modification of its sequence, or be completely replaced by more
active promoters even from heterologous organisms or of synthetic
origin.
The calpain inhibitors identified by the method according to the
invention are suitable for producing medicines for treating
disorders associated with calpain dysfunctions, for example
disorders selected from the group of cardiovascular,
immunological, inflammatory, allergic, neurological,
neurodegenerative or oncological diseases such as restenosis,
arthritis, ischemias of the heart, of the kidney or of the
central nervous system (eg. stroke), inflammations, muscular
dystrophies, cataracts in the eyes (eg. gray cataract), injuries
to the central nervous system (eg. trauma), Alzheimer's disease,
HIV-induced neuropathy, Parkinson's and Huntington's diseases,
preferably for producing medicines for treating disorders of the
placenta, such as gestosis, or of embryogenesis.
005~~48288 CA 02301815 2000'U2-23
l
The CAPN6 gene sequences according to the invention are also
advantageously suitable for diagnosing disorders or for gene
therapy.
Examples
Example 1: Cloning of the CAPN6 gene
The mouse CAPN6 sequence (EMBL accession number Y12583) was
cloned by the RACE method using tag 17 mouse embryos and primer
sequences derived from the sequence EST AA050030. A plasmid clone
which contained the corresponding EST sequences was obtained from
the I.M.A.G.E consortium (Research Genetics Inc.). The human
CAPN6 homolog was obtained by a search for homology in the EST
databank using the mouse protein sequence and the tblastn
algorithm. It was possible to combine the part-sequences which
were found to give an incomplete sequence of human CAPN6 with a
length of 1083 nucleotides (SEQ ID NO: 3).
ZO Example 2: Expression of the CAPN6 gene in various tissues
rr~,,xpression of the CAPN6 gene in various tissues was detected
using a 32P-labeled human cDNA fragment with a human RNA master
blot supplied by Clontech which contains RNA from 50 different
tissues. The hybridization and the highly stringent washing
conditions were carried out in accordance with the manufacturer's
instructions. The CAPN6 cDNA fragment used for the expression
experiments was a 2.2 kb EcoRI/XhoI fragment which comprises the
EST AA050030. CAPN6 was expressed only in placental tissue (see
Figure 4). As control, the blot was carried out with a human
ubiquitin DNA sample in order to determine the RNA loading.
3rd example: Location of the CAPN6 gene on the chromosome
The human gene was located using the NIGMS human/rodent somatic
cell hybrid "mapping panel" (Coriell Cell Repositories). The
primer sequences used for the PCR were as follows:
5'-gttgaaactgattggggtctg-3' and 5'-ctgtcttcccaaggggtttctc-3'. The
PCR amplification was carried out with an annealing temperature
of 58°C and resulted in a 200 by fragment. The results were
examined for agreement between the presence of human chromosomes
and the PCR product. The exact location of the gene in the human
chromosome was found using the Stanford G3 RH Panel (Research
Genetics) and transmission of the PCR results to the localization
service of the Stanford Human Genome Center
0050/48288 ca o23oisis 2000-02-23
19
(http://www-shgc.stanford.edu). The human CAPN6 gene was found on
the X chromosome coupled to the DXS7356 marker.
4th example: Cathepsin H test
Cathepsin B inhibition was determined by a method similar to that
of S. Hasnain et al., J. Biol. Chem. 268, 1993, 235 - 240.
2 ~1 of an inhibitor solution prepared from inhibitor and DMSO
(final concentration: 100 ~,M to 0.01 ~tM) are added to 88 ~,1 of
cathepsin B (cathepsin B from human liver supplied by Calbiochem,
diluted to 5 units in 500 ~tM buffer). This mixture is preincubated
at room temperature (= 25°C) for 60 minutes and then the reaction
is started by adding 10 ~.1 of 10 mM Z-Arg-Arg-pNA (in buffer with
10% DMSO). The reaction is followed in a microtiter plate reader
at 405 nm for 30 minutes. The ICSO values are then determined from
the maximum slopes.
5th example: Calpain test
The activity of the calpain inhibitors was investigated in~a
colorimetric test with Hammarsten casein (Merck, Darmstadt)
as substrate. The test was carried out in microtiter plates in
accordance with the publication by Buroker-Kilgore and Wang in
Anal. Biochem. 208, 1993, 387 - 392. The enzyme used was CAPN6
which was expressed in one of the systems described above and
then purified. The substances were incubated with the enzyme at
room temperature for 60 minutes, the concentration of the solvent
DMSO not exceeding 1%. After addition of the Bio-Rad color
reagent, the optical density was measured at 595 nm in the SLT
Easy Reader EAR 400. The 50% activity of the enzyme is evident
from the optical densities determined at the maximum activity of
the enzyme without inhibitors and the activity of the enzyme
without addition of calcium.
6th example: Platelet test to determine the cellular activity of
calpain inhibitors
The calpain-mediated degradation of proteins in platelets was
carried out as described by Zhaozhao Li et al., J. Med. Chem.,
36, 1993, 3472- 3480. Human platelets were isolated from fresh
sodium citrate blood from donors and adjusted to 10~ cells/ml in
buffer (5 mM Hepes, 140 mM NaCl and 1 mg/ml BSA, pH 7.3).
Platelets (0.1 ml) are [sic] preincubated in 1 ~.1 of various
concentrations of inhibitors (dissolved in DMSO) for 5 minutes.
Then calcium ionophor A 23187 (1 ~.M in the test) and calcium (5 mM
in the test) were added and a further incubation took place at
uu5ui4~s~ts~s ca o23oisis Zooo-o2-23
37°C for 5 minutes. After a centrifugation step, the platelets
were taken up in SDS-page sample buffer and boiled at 95°C for
5 minutes, and the proteins were fractionated in an 8% gel.
Breakdown of the two proteins actin-binding protein (= ABP) and
5 talin was followed by quantitative densitometry since after
addition of calcium and ionophore, these proteins disappeared,
and a new band in the region of 200 Kd molecular weight appeared.
The half-maximum enzyme activity is determined from this.
15
25
35
45
, 0050/48288 Ca o23o1s15 2000-02-23
21
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: BASF Aktiengesellschaft
(B) STREET: Carl Bosch Strasse
(C) CITY: Ludwigshafen
(D) STATE: Rheinland-Pfal2
(E) COUNTRY: Germany
(F) POSTAL CODE: D-67056
(ii) TITLE OF APPLICATION: Novel tissue-specific calpains,
their preparation and use
(iii) NUMBER OF SEQUENCES: ~
(iv) COMPUTER-READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version .#1.25 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i_) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2069 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii [sic]) ANTISENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(vii) IMMEDIATE SOURCE:
(B) CLONE: CAPN6
, 0050/48288 CA 02301815 2000-02-23
22
' (ix) FEATURES:
(A) NAME/KEY: 5'UTR
(B) LOCATION: 1..129
(ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 130..2055
(ix) FEATURES:
(A) NAME/KEY: 3'UTR
(B) LOCATION: 2056..2069
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
GGGGTTACCT GGCTAAGAGC AGCAGCAGCA GCAGCAGCAG CAGCAGCAGT AGCAGCAGCA 60
GCAGCAGCAG CAGCAGCAGC AGCAGCAGCA GCAGGGTTCC TGAGCTAACT CAGACCTAGT 120
TTGATAGCA ATG GGT CCT CCT CTG AAG CTC 'TTC AAei AAC CAG AAG TAC 168
Met Gly Pro 9ro Leu Lys Leu Phe Lys Asn Gln Lys Tyr
1 5 10
CAA GAA CTG AAG CAG GAG TGC ATG AAG GAT GGC CGC CTT TTC TGT GAC 216
Gln Glu Leu Lys Gln Glu Cys Met Lys Asp Gly Arg Leu Phe Cys Asp
15 20 25
CCA ACC TTC CTA CCG GAG AAT GAT TCT CTG TTT TTC AAC CGG CTG CTT 264
Pro Thr Phe Leu Pro Glu Asn Asp Ser Leu Phe Phe Asn Arg Leu Leu
30 35 40 45
CCT GGG AAG GTG GTG TGG AAG CGT CCA CAG GAC ATT TCT GAT GAC CCC 312
Pro Gly Lys Val Val Trp Lys Arg Pro Gln Asp Ile Ser Asp Asp Pro
50 55 60
CAC CTG ATT GTG GGC AAC ATC AGC AAC CAC CAG CTG ATC CAG GGC AGA 360
His Leu Ile Val Gly Asn Ile Ser Asn His Gln Leu Ile Gln Gly Arg
65 70 75
TTG GGG AAC AAG GCA ATG ATC TCT GCA TTT TCC TGT TTG GCT GTT CAG 408
Leu Gly Asn Lys Ala Met Ile Ser Ala Phe Ser Cys Leu Ala Val Gli
80 85 90
,~ U05U/48288 CA 02301815 2000-02-23
23
GAG TCA CAC TGG ACA AAG GCA ATT CCC AAC CAC AAG GAT CAG GAA TGG 456
Glu Ser His Trp Thr Lys Ala Ile Pro Asn His Lys Asp Gln Glu Trp
95 1Q0 105
GAT CCT CGA AAG CCA GAG AAA TAC GCT GGA ATC TTT CAC TTC CGC TTC 504
Asp Pro Arg Lys Pro Glu Lys Tyr Ala Gly Ile Phe His Phe Arg Phe
110 115 120 125
TGG CAT TTT GGA GAA TGG ACC GAG GTG GTG ATT GAT GAC TTG CTT CCC 552
Trp His Phe Gly Glu Trp Thr Glu Val Val Ile Asp Asp Leu Leu Pro
130 135 140
ACC ATC AAC GGA GAT CTG GTC TTC TCA TTC TCC ACC TCC ATG AAT GAG 600
Thr Ile Asn Gly Asp Leu Val Phe Ser Phe Ser Thr Ser Met Asn Glu
145 150 155
TTT TGG AAT GCT CTA CTG GAA AAA GCG TAT GCA AAG CTG CTG GGC TGT 648
Phe Trp Asn Ala Leu Leu Glu Lys Ala Tyr Ala Lys Leu Leu Gly Cys
160 165 I7U
TAT GAG GCT TTG GAT GGT CTG ACC ATC ACT GAT ATC ATC ATG GAC TTC 696
Tyr Glu .Ala Leu Asp Gly Leu Thr Ile Thr Asp Ile Ile Met Asp Phe
175 180 185
ACT GGC ACA CTG GCT GAA ATC ATT GAC ATG CAG AAA GGA CGA TAC ACT 744
Thr Gly Thr Leu Ala Glu Ile Ile Asp Met Gln Lys Gly Arg Tyr Thr
I90 195 200 205
GAT CTT GTT GAG GAG AAG TAC AAG CTG TTT GGA GAA CTG TAC AAA ACG 792
Asp Leu Val Glu Glu Lys Tyr Lys Leu Phe Gly Glu Leu Tyr Lys Thr
210 215 220
TTC ACC AAA GGA GGT CTA ATT TGC TGC TCC ATT GAG TCT CCC AGC CAG 840
Phe Thr Lys Gly Gly Leu Ile Cys Cys Ser Ile Glu Ser Pro Ser Glr
225 230 235
GAG GAA CAA GAA GTT GAA ACA GAC TGG GGA CTA CTG AAG GGT TAT ACC 888
Glu Glu Gln Glu Val Glu Thr Asp Trp Gly Leu Leu Lys Gly Tyr Thr
240 245 250
TAC ACC ATG ACT GAT ATT CGC AAG CTC CGT CTC GGA GAA AGA CTT GTG 936
Tyr Thr Met Thr Asp Ile Arg Lys Leu Arg Leu Gly Glu Arg Leu Val
255 260 265
0050/48288 Ca o23o1s15 2000-02-23
24
GAA GTC TTC AGT ACT GAG AAG CTG TAT ATG GTT CGC CTA AGG AAC CCA 984
Glu Val Phe Ser Thr Glu Lys Leu Tyr Met Val Arg Leu Arg Asn Pro
270 275 280 285
TTG GGA AGA CAG GAA TGG AGT GGC CCC TGG AGT GAA ATT TCA GAG GAG 1032
Leu Gly Arg Gln Glu Trp Ser Gly Pro Trp Ser Glu Ile Ser Glu Glu
290 295 300
TGG CAG CAA CTG ACT GTA ACA GAT CGC AAG AAC CTA GGA CTT GTT ATG 1080
Trp Gln Gln Leu Thr Val Thr Asp Arg Lys Asn Leu Gly Leu Val Met
305 310 315
TCT GAT GAT GGA GAA TTT TGG ATG AGT CTG GAA GAT TTT TGC CAC AAC 1128
Ser Asp Asp Gly Glu Phe Trp Met Ser Leu Glu Asp Phe Cys His Asn
320 325 330
TTT CAC AAA CTG AAT GTC TGC CGC AAT GTG AAT AAT CCT GTT TTT GGC 1176
Phe His Lys Leu Asn Val Cys Arg Asn Val Asn Asn Pro Val Phe Gly
335 340 345
CGC AAG GAG CTG GAA TCA GTG GTG GGi1 TGT TGG ACT GTG GAT GAT GAC 1224
Arg Lys Glu Leu Glu Ser Val Val Gly Cys Trp Thr Val Asp Asp Asp
35C 355 360 365
CCT C'PG ATG AAC CGA TCA GGA GGT TGC TAT AAC AAC CGT GAT ACC TTC 1272
Pro LeL Met Asr. Arg Ser Gly Gly Cys Tyr Asn Asn Arg Asp Thr PhE
370 375 380
TTG CAG AAT CCT CAG TAC ATT TTC ACT GTG CCC GAG GAT GGC CAT AAA 1320
Leu.Gln Asn Pro Gln Tyr Ile Phe Thr Val Pro Glu Asp Gly His Lys
385 390 395
GTC ATC ATG TCA CTG CAA CAG AAG GAC CTA CGC ACT TAC CGC CGA ATG 1368
Val Ile Met Ser Leu Gln Gln Lys Asp Leu Arg Thr Tyr Arg Arg Met
400 405 410
GGA AGA CCT GAT AAT TAC ATC ATT GGT TTT GAG CTC TTC AAG GTG GAG 1416
Gly Arg Pro Asp Asn Tyr Ile Ile Gly Phe Glu Leu Phe Lys Val Glu
415 420 425
ATG AAC CGA AGG TTC CGT CTT CAC CAT CTG TAT ATT CAG GAG CGT GCT 1464
Met Asn Arg Arg Phe Arg Leu His His Leu Tyr Ile Gln Glu Arg Ala
430 435 440 445
uu~vi ~eo~oo CA 02301815 2000-02-23
GGG ACT TCC ACT TAT ATC GAC ACC CGT ACT GTG TTT CTG AGC AAG TAT 1512
Gly Thr Ser Thr Tyr Ile Asp Thr Arg Thr Val Phe Leu Ser Lys Tyr
450 455 460
CTG .AAG AAG GGC AGC TAC GTG CTT GTT CCA ACC ATG TTC CAA CAT GGC 1560
Leu Lys Lys Gly Ser Tyr Val Leu Val Pro Thr Met Phe Gln His Gly
465 470 475
CGT ACC AGT GAA TTT CTG CTG AGG ATC TTC TCT GAA GTG CCC GTC CAG 1608
Arg Thr Ser Glu Phe Leu Leu Arg Ile Phe Ser Glu Val Pro Val Gln
480 485 490
CTC AGG GAA CTG ACC TTG GAC ATG CCC AAG ATG TCT TGC TGG AAC CTG 1656
Leu Arg Glu Leu Thr Leu Asp Met Pro Lys Met Ser Cys Trp Asn Leu
495 500 505
GCA CGT GGC TAC CCA AAG GTG GTT ACC CAG ATC ACT GTC CAC AGT GCT 1704
Ala Arg Gly Tyr Pro Lys Val Val Thr !;ln Ile Thr Val His Ser Ala
510 515 520 525
GAG GGC CTG GAG AAG AAG TAT'GCC AAT GAA ACT GTC AAT CCA TAT CTG ~ 175'1.
Glu Gly Leu Glu Lys Lys Tyr Ala Asn Glu Thr Val Asn Pro Tyr Leu
530 535 540
ATC ATC AAA TGT GGA AAG GAG GAA GTC CGT TCC CCT GTC CAG AAG AAT 1800
Ile Ile Lys Cys Gly Lys Glu Glu Val Arg Ser Pro Val Gln Lys Asn.
545 550 555
ACT GTG CAT GCC ATT TTT GAC ACG CAG GCC GTT TTC TAC AGA AGG ACC 1848
Thr Val His Ala Ile Phe Asp Thr Gln Ala Val Phe.Tyr Arg Arg Thr
560 565 570
ACT GAC ATT CCT ATT ATC ATC CAG GTG TGG AAC AGC AGA AAA TTC TGT 1896
Thr Asp Ile Pro Ile Ile Ile Gln Val Trp Asn Ser Arg Lys Phe Cys
575 580 585
GAT CAG TTC CTG GGG CAG GTT ACT CTC GAT GCT GAC CCC AGC GAC TGC 1944
Asp Gln Phe Leu Gly Gln Val Thr Leu Asp Ala Asp Pro Ser Asp Cys
590 595 600 605
CGT GAT CTG AAA TCT CTG TAC CTG CGT AAG AAG GGT GGT CCT ACT GCC 1992
Arg Asp Leu Lys Ser Leu Tyr Leu Arg Lys Lys Gly Gly Pro Thr Ala
610 615 620
v~rrv~ Seo'oo CA 02301815 2000-02-23
26
AAA GTC AAG CAA GGT CAC ATC AGC TTC AAA GTT ATC TCT AGC GAT GAT 2040
Lys Val Lys Gln Gly His Ile Ser Phe Lys Val Ile Ser Ser Asp Asp
625 630 635
CTC ACT GAG CTC TAAGTAGTCA TCATCAG 2069
Leu Thr Glu Leu
640
(2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 641 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:
P2et Gly Pro Pro Leu Lys Leu Phe Lys Asn Gln Lys Tyr Gln Glu Leu
1 5 10 15
Lys Gln Glu Cys Met Lys App Gly Arg Leu Phe Cys Asp Pro Thr Phe
20 25 30
Leu Pro Glu Asn Asp Ser Leu Phe Phe Asn Arg Leu Leu Pro Gly Lys
35 40 45
Val Val Trp Lys Arg Pro Gln Asp Ile Ser Asp Asp Pro His Leu Ile
50 55 60
Val Gly Asn Ile Ser Asn His Gln Leu Ile Gln Gly Arg Leu Gly Asn
65 70 75 80
Lys Ala Met Ile Ser Ala Phe Ser Cys Leu Ala Val Gln Glu Ser His
85 90 95
Trp Thr Lys Ala Ile Pro Asn His Lys Asp Gln Glu Trp Asp Pro Arg
100 105 110
Lys Pro Glu Lys Tyr Ala Gly Ile Phe His Phe Arg Phe Trp His Phe
115 120 125
VV:JVI'sos.GO CA 02301815 2000-02-23
27
Gly Glu Trp Thr Glu Val Val Ile Asp Asp Leu Leu Pro Thr Ile Asn
130 135 140
Gly Asp Leu Val Phe Ser Phe Ser Thr Ser Met Asn Glu Phe Trp Asn
145 150 155 160
Ala Leu Leu Glu Lys Ala Tyr Ala Lys Leu Leu Gly Cys Tyr Glu Ala
165 170 175
Leu Asp Gly Leu Thr Ile Thr Asp Ile Ile Met Asp Phe Thr Gly Thr
180 185 190 -
Leu Ala Glu Ile Ile Asp Met Gln Lys Gly Arg Tyr Thr Asp Leu Val
195 200 205
Glu G1u Lys Tyr Lys Leu Phe Gly Glu Leu Tyr Lys Thr Phe Thr Lys
210 215 220
Gly Gly Leu Ile Cys Cys Ser Ile Glu Ser Pro Ser Gln Glu Glu Gln
225 230 235 240
Glu Val Glu Thr Asp Trp Gly Leu Leu Lys Gly Tyr Thr Tyr Thr Met
245 250 255
Thr Asp Ile Arg Lys Leu Arg Leu Gly Glu Arg Leu Val Glu Val Phe
260 265 270
Ser Thr Glu Lys Leu Tyr Met Val Arg Leu Arg Asn Pro Leu Gly Arg
275 280 285
Gln Glu Trp Ser Gly Pro Trp Ser Glu Ile Ser Glu Glu Trp Gln Gln
290 295 ~ 300
Leu Thr Val Thr Asp Arg Lys Asn Leu Gly Leu Val Met Ser Asp Asp
305 310 315 320
Gly Glu Phe Trp Met Ser Leu Glu Asp Phe Cys His Asn Phe His Lys
325 330 335
Leu Asn Val Cys Arg Asn Val Asn Asn Pro. Val Phe Gly Arg Lys Glu
340 345 350
vv:rvi 5eo~.ov CA 02301815 2000-02-23
28
Leu Glu Ser Val Val Gly Cys Trp Thr Val Asp Asp Asp Pro Leu Met
355 360 365
Asn Arg Ser Gly Gly Cys Tyr Asn Asn Arg Asp Thr Phe Leu Gln Asn
370 375 380
Pro Gln Tyr Ile Phe Thr Val Pro Glu Asp Gly His Lys Val Ile Met
385 390 395 400
Ser Leu Gln Gln Lys Asp Leu Arg Thr Tyr Arg Arg Met Gly Arg Pro
405 410 415
Asp Asn Tyr Ile Ile Gly Phe Glu Leu Phe Lys Val Glu Met Asn Arg
420 425 430
Arg Phe Arg Leu His His Leu Tyr Ile Gln Glu Arg Ala Gly Thr Ser
435 440 445
Thr Tyr Ile Asp Thr Arg Thr Val Phe Leu Ser Lys Tyr Leu Ly5 Lys
450 455 460
Gly Ser Tyr Val Leu Val Pro Thr Met Phe Gln His Gly Arg Thr Ser
465 470 475 480
Glu Phe Leu Leu Arg Ile Phe Ser Glu Val Pro Val Gln Leu Arg Glu
485 490 495
Leu Thr Leu Asp Met Pro Lys Met Ser Cys Trp Asn Leu Ala Arg Gly
500 505 510
Tyr Pro Lys Val Val Thr Gln Ile Thr Val His Ser Ala Glu Gly Leu
515 520 525
Glu Lys Lys Tyr Ala Asn Glu Thr Val Asn Pro Tyr Leu Ile Ile Lys
530 535 540
Cys Gly Lys Glu Glu Val Arg Ser Pro Val Gln Lys Asn Thr Val His
545 550 555 560
Ala Ile Phe Asp Thr Gln Ala Val Phe Tyr Arg Arg Thr Thr Asp Ile
565 570 575
vv:rm =u~.uo CA 02301815 2000-02-23
29
Pro Ile Ile Ile Gln Val Trp Asn Ser Arg Lys Phe Cys Asp Gln Phe
580 585 590
Leu Gly Gln Val Thr Leu Asp Ala Asp Pro Ser Asp Cys Arg Asp Leu
595 600 605
Lys Ser Leu Tyr Leu Arg Lys Lys Gly Gly Pro Thr Ala Lys Val Lys
610 615 620
Gln G1y His Ile Ser Phe Lys Val Ile Ser Ser Asp Asp Leu Thr Glu
625 630 635 640
Leu
(2) INFORMATION FOR SEQ ID NO: 3:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1125 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii [sic]) ANTISENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(vii) IMMEDIATE SOURCE:
(B) CLONE: CAPN6
(ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 2..1125
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
vvavi ~so~oo CA 02301815 2000-02-23
G CTT GTT GAG GAG AAG TAC AAG CTA TTC GGA GAA CTG TAC AAA ACA 46
Leu Val Glu Glu Lys Tyr Lys Leu Phe Gly Glu Leu Tyr Lys Thr
1 5 10 15
TTT ACC AAA GGT GGT CTG ATC TGC TGT TCC ATT GAG TCT CCC AAT CAG 94
Phe Thr Lys Gly Gly Leu Ile Cys Cys Ser Ile Glu Ser Pro Asn Gln
20 25 30
GAG GAG CAA GAA GTT GAA ACT GAT TGG GGT CTG CTG AAG GGC CAT ACC 142
Glu Glu Gln Glu Val Glu Thr Asp Trp Gly Leu Leu Lys Gly His Thr
40 45
TAT ACC ATG ACT GAT ATT CGC AAA ATT CGT CTT GGA GAG AGA CTT GTG 190
Tyr Thr Met Thr Asp Ile Arg Lys Ile Arg Leu Gly Glu Arg Leu Val
50 55 60
GAA GTC TTC AGT GCT GAG AAG CTG TAT ATG GTT CGC CTG AGA AAC CCC 238
Gln Val Phe Ser Ala Glu Lys Leu Tyr Met Val Arg Leu Arg Asn Pro
55 '70 75
TTG GGA AGA CAG GAA TGG AGT GGC CCC TGG AGT GAA ATT TCT GAA GAG 286
Leu G1y Arg Gln Glu Trp Ser Gly Pro Trp Ser Glu Ile Ser Glu Glu
80 85 90 95
TGG CAG CAA CTG ACT GCA TCA GAT CGC AAG AAC CTG GGG CTT G'tT ATG 334
Trp Gln Gln Leu Thr Ala Ser Asp Arg Lys Asn Leu Gly Leu Val Met
100 105 110
TCT GAT GAT GGA GAG TTT TGG ATG AGC TTG GAG GAC TTT TGC CGC AAC 382
Ser Asp Asp Gly Glu Phe Trp Met Ser Leu Glu Asp Phe Cys Arg Asn
115 120 125
TTT CAC AAA CTG AAT GTC TGC CGC AAT GTG AAC AAC CCT ATT TTT GGC 430
Phe His Lys Leu Asn Val Cys Arg Asn Val Asn Asn Pro Ile Phe Gly
130 135 140
CGA AAG GAG CTG GAA TCG GTG TTG GGA TGC TGG ACT GTG GAT GAT GAT 478
Arg Lys Glu Leu Glu Ser Val Leu Gly Cys Trp Thr Val Asp Asp Asp
145 150 155
CCC CTG ATG AAC CGC TCA GGA GGC TGC TAT AAC AAC CGT GAT ACC TTC 526
Pro Leu Met Asn Arg Ser Gly Gly Cys Tyr Asn Asn Arg Asp Thr Phe
160 165 170 175
,.
vv~vi,eoaoo CA 02301815 2000-02-23
31
CTG CAG AAT CCC CAG TAC ATC TTC ACT GTG CCT GAG GAT GGG CAC AAG 574
Leu Gln Asn Pro Gln Tyr Ile Phe Thr Val Pro Glu Asp Gly His Lys
180 185 190
GTC ATT ATG TCA CTG CAG CAG AAG GAC CTG CGC ACT TAC CGC CGA ATG 622
Val Ile Met Ser Leu Gln Gln Lys Asp Leu Arg Thr Tyr Arg Arg Met
195 200 205
GGA AGA CCT GAC AAT TAC ATC ATT GGC TTT GAG CTC TTC AAG GTG GAG 670
Gly Arg Pro Asp Asn Tyr Ile Ile Gly Phe Glu Leu Phe Lys Val Glu
210 215 220
ATG AAC CGC AAA TTC CGC CTC CAC CAC CTC TAC ATC CAG GAG CGT GCT 718
Met Asn Arg Lys Phe Arg Leu His His Leu Tyr Ile Gln Glu Arg Ala
225 230 235
GGG ACT TCC ACC TAT ATT GAC ACC CGC ACA GTG TTT CTG AGC AAG TAC 766
Gly Thr Ser Thr Tyr Ile Asp Thr Arg Thr Val Phe L,eu Ser Lys Tyr
240 245 250 255
CTG AAG AAG GGC AAC TAT GTG CTT GTC CCA ACC ATG TTC CAG CAT GGT 814
~eu Lys ~ys Gly Asn Tyr Val Leu Val Pro Thr rlet Phe Gln His Gly
260 265 ~70
CGC ACC AGC GAG TTT CTC CTG AGA ATC TTC TCT GAA GTG CCT GTC CAG 862
erg Thr Ser Glu Phe Leu Leu Arg Ile Phe Ser Glu Val Pro Val Gln
275 280 285
CTC AGG GAA CTG ACT CTG GAC ATG CCC AAA ATG TCC TGC TGG AAC CTG 910
Leu Arg Glu Leu Thr Leu Asp Met Pro Lys Met Ser Cys Trp Asn Leu
290 295 300
GCT CGT GGC TAC CCG AAA GTA GTT ACT CAG ATC ACT GTT CAC AGT GCT 958
Ala Arg Gly Tyr Pro Lys Val Val Thr Gln Ile Thr Val His Ser Ala
305 310 315
GAG GAC CTG GAG AGG AGG TAT GCC AAT GGA ACT GTA AAC CCA TAT TTG 1006
Glu Asp Leu Glu Arg Arg Tyr Ala Asn Gly Thr Val Asn Pro Tyr Leu
320 325 330 335
GTC ATC AAA TGT GGA AAG GAG GAA GTC CGT TCT CCT GTC CAG AAA AAT 1054
Val Ile Lys Cys Gly Lys Glu Glu Val Arg Ser Pro Val Gln Lys Asn
340 345 350
""'"'~ "°'°° CA 02301815 2000-02-23
32
ACA GTT CAT GCC ATT TTT GAC ACC CAT GCC ATT TTC TAC AGA AGG ACC 1102
Thr Val His Ala Ile Phe Asp Thr His Ala Ile Phe Tyr Arg Arg Thr
355 360 365
ACG GAC ATT CCT ATT ATA GTA CA 1125
Thr Asp Ile Pro Ile Ile Val
370
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
Leu Val Glu Glu Lys Tyr Lys Leu Phe Gly Glu Leu Tyr Lys Thr Phe
1 5 10 15
Thr Lys Gly Gly Leu Ile Cys Cys Ser Ile Glu Ser Pro Asn Gln Glu
20 25 30
Glu Gln Glu Val Glu Thr Asp Trp Gly Leu Leu Lys Gly His Thr Tyr
35 40 45
Thr Met Thr Asp Ile Arg Lys Ile Arg Leu Gly Glu Arg Leu Val Glu
50 55 60
Val Phe Ser Ala Glu Lys Leu Tyr Met Val Arg Leu Arg Asn Pro Leu
65 70 75 80
Gly Arg Gln Glu Trp Ser Gly Pro Trp Ser Glu Ile Ser Glu Glu Trp
85 90 95
Gln Gln Leu Thr Ala Ser Asp Arg Lys Asn Leu Gly Leu Val Met Ser
100 105 110
Asp Asp Gly Glu Phe Trp Met Ser Leu Glu Asp Phe Cys Arg Asn Phe
115 120 125
VVJV/ 'i0600 CA 02301815 2000-02-23
33
His Lys Leu Asn Val Cys Arg Asn Val Asn Asn Pro Ile Phe Gly Arg
130 135 140
Lys Glu Leu Glu Ser Val Leu Gly Cys Trp Thr Val Asp Asp Asp Pro
145 150 155 160
Leu Met Asn Arg Ser Gly Gly Cys Tyr Asn Asn Arg Asp Thr Phe Leu
165 170 175
Gln Asn Pro Gln Tyr Ile Phe Thr Val Pro Glu Asp Gly His Lys Val
180 185 190
Ile Met Ser Leu Gln Gln Lys Asp Leu Arg Thr Tyr Arg Arg Met Gly
195 200 205
Arg Pro. Asp Asn Tyr Ile Ile Gly Phe Glu Leu Phe Lys Val Glu Met
210 215 220
Asn Arg Lys Phe Arg Leu His His Leu Tyr Ile Gln Glu Arg Ala Gly
225 230 235 240
Thr Ser Thr Tyr Ile Asp Thr Arg Thr Val Phe Leu Ser Lys Tyr Leu
245 250 255
Lys Lys Gly Asn Tyr Val Leu Val Pro Thr Met Phe Gln His Gly Arg
260 265 270
Thr Ser Glu Phe Leu Leu Arg Ile Phe Ser Glu Val Pro Val Gln Leu
275 280 285
Arg Glu Leu Thr Leu Asp Met Pro Lys Met Ser Cys Trp Asn Leu Ala
290 295 300
Arg Gly Tyr Pro Lys Val Val Thr Gln Ile Thr Val His Ser Ala Glu
305 310 315 320
Asp Leu Glu Arg Arg Tyr Ala Asn Gly Thr Val Asn Pro Tyr Leu Val
325 330 335
Ile Lys Cys Gly Lys Glu Glu Val Arg Ser Pro Val Gln Lys Asn Thr
340 345 350
vv;rvi 5eo~.oo CA 02301815 2000-02-23
34
Val His Ala Ile Phe Asp Thr His Ala Ile Phe Tyr Arg Arg Thr Thr
355 360 365
Asp Ile Pro Ile Ile Val
370
