U.S. patent application number 09/871388 was filed with the patent office on 2002-09-12 for kuz, a novel family of metalloproteases.
Invention is credited to Pan, Duojia, Rooke, Jenny, Rubin, Gerald M., Xu, Tian, Yavari, Reza.
Application Number | 20020127621 09/871388 |
Document ID | / |
Family ID | 26692174 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020127621 |
Kind Code |
A1 |
Rubin, Gerald M. ; et
al. |
September 12, 2002 |
KUZ, a novel family of metalloproteases
Abstract
Members of a novel family of polypeptides, the KUZ family, are
metalloproteases involved in neuronal partitioning and neuronal
development. The invention provides KUZ poylpeptides, antibodies
that bind the KUZ polypeptides, KUZ encoding nucleic acids, methods
for identifying cells expressing the KUZ polypeptides, methods of
identifying ligands that bind to the subject proteins and methods
of blocking KUZ polypeptide/ligand interactions.
Inventors: |
Rubin, Gerald M.; (Berkeley,
CA) ; Pan, Duojia; (Berkeley, CA) ; Rooke,
Jenny; (New Haven, CT) ; Yavari, Reza; (New
Haven, CT) ; Xu, Tian; (New Haven, CT) |
Correspondence
Address: |
RICHARD ARON OSMAN
SCIENCE AND TECHNOLOGY LAW GROUP
75 DENISE DRIVE
HILLSBOROUGH
CA
94010
|
Family ID: |
26692174 |
Appl. No.: |
09/871388 |
Filed: |
May 31, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09871388 |
May 31, 2001 |
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09709126 |
Nov 8, 2000 |
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09709126 |
Nov 8, 2000 |
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09285502 |
Apr 2, 1999 |
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09285502 |
Apr 2, 1999 |
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08937931 |
Aug 27, 1997 |
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60053476 |
Jul 23, 1997 |
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60019390 |
Aug 29, 1996 |
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Current U.S.
Class: |
435/7.32 ;
435/183; 435/226 |
Current CPC
Class: |
C12N 9/6489 20130101;
C12Q 1/37 20130101; C12N 9/6402 20130101; G01N 33/573 20130101 |
Class at
Publication: |
435/7.32 ;
435/183; 435/226 |
International
Class: |
G01N 033/554; G01N
033/569; C12N 009/64 |
Claims
What is claimed:
1. An isolated KUZ polypeptide comprising a sequence selected from
SEQ ID NOS:2, 4, 6, 8 and 10 or a polypeptide domain thereof having
at least 15 consecutive residues thereof and at least one
KUZ-specific activity selected a KUZ-specific antigenicity and a
KUZ- specific immunogenicity.
2. An isolated KUZ polypeptide made by a method comprising the
following steps: incubating a host cell or cellular extract
containing a recombinant nucleic acid encoding a polypeptide
according to claim 1. under conditions whereby the polypeptide
encoded by the nucleic acid is expressed and recovering the
expressed polypeptide.
3. An isolated KUZ polypeptide encoded by a first nucleic acid
specifically hybridizable to a second nucleic acid having a
sequence defmed by the corresponding opposite strand of
SEQIDNOS:1,3,5,7or9.
4. A method of screening for an agent which modulates the binding
of a KUZ polypeptide to a binding target, said method comprising
the steps of: contacting a polypeptide according to claim 1 with a
binding target of said polypeptide in the presence of a candidate
agent, and detecting or measuring the binding of the polypeptide to
said binding target, wherein a difference in the amount of said
binding relative to the amount of binding in the absence of the
candidate agent indicates that the agent modulates the binding of
said polypeptide to said binding target.
5. A method of screening for an agent which modulates the cleavage
of a Notch protein by a KUZ polypeptide, said method comprising the
steps of: contacting a polypeptide according to claim 1 with a
Notch protein in the presence of a candidate agent, and detecting
or measuring the amount of Notch protein cleavage products thereby
produced, wherein a difference in the identities or amount of Notch
protein cleavage products thus produced relative to the identities
or amount of said products in the absence of the candidate agent
indicates that the agent modulates the cleavage of the Notch
protein by the KUZ polypeptide.
6. A method for modulating the interaction of a KUZ polypeptide
according to claim 1 with a natural KUZ binding target comprising
the step of exposing said polypeptide or ,,aid binding target to an
agent that modulates the binding of said polypeptide to said
binding target.
7. A method according to claim 6, wherein (i) said binding target
is a Notch protein andlor (ii) said agent is selected from a
KUZ-specific antibody, a dominant negative fragment of a KUZ
polypeptide and a metalloprotease inhibitor.
8. A polypeptide according to claim 1, which is a dominant-negative
mutant of a KUZ polypeptide.
9. A method for modulating the Notch signal transduction pathway in
a cell comprising providing the cell with an agent which modulates
activity of a KUZ polypeptide or function of a kuz gene, in which
the agent is a polypeptide according to claim 1 provided to the
cell by (i) intracellular expression from a recombinant nucleic
acid or (ii) exogenous contacting of the cell.
10. An isolated deriviative of the polypeptide of claim 1, wherein
one or more conservative amino acid substitutions have been made in
said sequence or consecutive residues and said derivative has at
least one of: one or more ftnctional activities of a KUZ protein;
one or more insertions, substitutions or deletions; and an ability
to be secreted from a cell.
11. An isolated chimeric polypeptide comprising at least 15
contiguous amino acids of a KUZ polypeptide sequence joined to an
amino acid sequence of a polypeptide other than a KUZ
polypeptide.
12. A method for determining the effect of a candidate drug on a
host deficient in KUZ polypeptide function comprising contacting a
host deficient in KUZ polypeptide function with a candidate drug;
and detecting the presence or absence of a physiological change in
said host in response to the contacting of said candidate drug,
wherein the candidate drug is a KUZ polypeptide according to claim
1.
13. The method of claim 12, wherein the host is a transgenic animal
having at least one disrupted kuz allele.
Description
FIELD OF THE INVENTION
[0001] The field of the invention is a novel family of proteins and
genes involved in development.
BACKGROUND OF THE INVENTION
[0002] Cell-cell interactions play an important role in regulating
cell fate decisions and pattern formation during the development of
multicellular organisms. One of the evolutionarily conserved
pathways that plays a central role in local cell interactions is
mediated by the transmembrane receptors encoded by the Notch (N)
gene of Drosophila, the lin-12 and glp-1 genes of C. elegans, and
their vertebrate homologs (reviewed in Artavanis-Tsakonas, S., et
al. (1995) Notch Signaling. Science 268, 225-232). collectively
hereinafter referred to as NOTCH receptors. Several lines of
evidence suggest that the proteolytic processing of NOTCH receptors
is mportant for their function. For example, in addition to the
full length proteins, antibodies against the intracellular domains
of NOTCH receptors have detected C- terminal fragments of 100-120
kd (hereafter referred to as 100 kd fragments); see e.g. Fehon, R.
G., et al. (1990). Cell 61, 523-534; Crittenden, S. L., et al.
(1994). Development 120, 2901-2911; Aster, J., et al. (1994) Cold
Spring Harbor Symp. Quart. Biol. 59., 125-136; Zagouras, P., et
al.(1995). Proc. Natl. Acad. Sci. USA 92, 6414-6418; and Kopan, R.,
et al. (1996). Proc. Natl. Acad. Sci. USA 93, 1683-1688. However,
the mechanism(s) of NOTCH activation have been hitherto largely
unknown.
[0003] During neurogenesis, a single neural precursor is singled
out from a group of equivalent cells through a lateral inhibition
process in which the emerging neural precursor cell prevents its
neighbors from taking on the same fate (reviewed in Simpson, P.
(1990). Development 109, 509-519). Genetic studies in Drosophila
have implicated a group of "neurogenic genes" including N in
lateral inhibition. Loss-of-function mutations in any of the
neurogenic genes result in hypertrophy of neural cells at the
expense of epidermis (reviewed in Campos-Ortega, J. A. (1993) In:
The Development ofDrosophila melanogaster M. Bate and A.
Martinez-Arias, eds. pp. 1091-1129. Cold Spring Harbor Press.). We
disclose herein a new neurogenic gene family, kuzbanian (kuz)
(Rooke, J., Pan, D. J., Xu, T. and Rubin, J. M. (1996). Science
273, 1227-1231). Members of the disclosed KUZ family of proteins
are shown to belong to the recently defined ADAM family of
transmembrane proteins, members of which contain both a disintegrin
and metalloprotease domain (reviewed in Wolfsberg, T.
[0004] G., et al. (1995). J. Cell Biol. 131, 275-278, see also
Blobel, C. P., et al. (1992). Nature 356, 248-252, 1992;
Yagami-Hiromasa, T., et al. (1995). Nature 377, 652-656; Black, R.
A.., et al. (1997). Nature 385, 729-733, 1997; and Moss, M. L., et
al. (1997). Nature 385, 733-7316).
[0005] We further disclose herein various engineered mutant forms
of native KUZ proteins and their activities. We show that mutant
KUZ proteins lacking protease activity interfere with endogenous
KUZ activity and function as dominant negatives (indicating that
the protease activity of native KUZ is essential to its biological
functions) and that dominant negatives can perturb lateral
inhibition during neurogenesis and result in the overproduction of
primary neurons. We also show that proteolytic processing of NOTCH
in embryos to generate the 100 kd species fails to occur in the kuz
mutant embryo and expression of dominant negatives in imaginal
discs or tissue culture cells blocks NOTCH processing (indicating
that the primary NOTCH translation product is proteolytically
cleaved by native KUZ proteins as part of the normal biosynthesis
of a functional NOTCH receptor).
SUMMARY OF THE INVENTION
[0006] The invention provides methods and compositions relating to
isolated KUZ polypeptides, related nucleic acids, polypeptide
domains thereof having KUZ-specific structure and activity and
modulators of KUZ function, particularly Notch protease
activity.
[0007] KUZ polypeptides, nucleic acids and modulators thereof
regulate Notch signal transduction pathways and hence provide
important regulators of cell function. The polypeptides may be
produced recombinantly from transformed host cells from the subject
KUZ polypeptide encoding nucleic acids or purified from mammalian
cells. The invention provides isolated KUZ hybridization probes and
primers capable of specifically hybridizing with the disclosed KUZ
genes, KUZ-specific binding agents such as specific antibodies, and
methods of making and using the subject compositions in diagnosis
(e.g. genetic hybridization screens for K;UZ transcripts), therapy
(e.g. KUZ protease inhibitors to modulate Notch signal
transduction) and in the biopharmaceutical industry (e.g. as
immunogens, reagents for isolating additional natural kuz alleles,
reagents for screening bio/chemical libraries for ligands and lead
ancLior pharmacologically active agents, etc.).
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 (A). Sequence alignment of predicted KUZ proteins
from Drosophila (DKUZ), mouse (MKUZ) and Xenopus (XKUZ). The full
length amino acid sequence of MKUZ was deduced from the nucleotide
sequence of two overlapping cDNA clones. Partial amino acid
sequence of XKUZ was deduced from the nucleotide sequence of a PCR
product that includes parts of the disintegrin and Cys-rich
domains. The alignments were produced using Geneworks software
(IntelliGenetics). Residues identical among two species are
highlighted. Predicted finctional domains are indicated. Amino acid
sequences from which degenerate PCR primers were designed are
indicated with arrows. Orthologs of kuz are also present in C.
elegans (GenBank accession nos. D68061 and M79534), rat (Z48444),
bovine (Z21961) and human (Z48579).
[0009] FIG. 1 (B). Summary of constructs used in this study and
their overexpression phenotypes. Different domains are indicated by
shadings. Asterisks indicate where point mutations were introduced.
Constructs 1-9 are based on DKUZ, while MKUZDN is based on MKUZ.
Abbreviations: ++, strong phenotype; +, weak phenotype; 0, no
phenotype.
[0010] FIG. 1 (C). Schematic diagram of DKUZ, MKUZ and XKUZ. The
percentages given refer to sequence identity in the indicated
domains between MKUZ and either DKUZ or XKUZ.
[0011] FIG. 2 shows a schematic of how KUZ protease can process
NOTCH on the extracellular domain to generate an N- terminal
extracellular fragment and the C-tenninal 100 kd fragment
containing the transmembrane and the cytoplasmic domain. These two
fragments may remain tethered together to function as a competent
NOTCH receptor, analogous to the maturation of the SEVENLESS
receptor (Simon et al., 1989).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0012] The present invention provides isolated KUZ polypeptides,
isolated from a wide variety of sources including Drosophila,
human, mouse and Xenopus, as well as allelic variants, naturally
occurring and altered secreted forms, deletion mutants having KUZ-
specific sequence and/or bioactivity and mutants comprising
conservative amino acid substitutions. SEQ ID NOS: 1, 3, 5, 7 and 9
depict exemplary natural cDNAs encoding Drosphila, human
transmembrane, human soluble (lacking a transmembrane domain),
mouse and Xenopus members, respectively, of the disclosed KUZ
family. SEQ ID NOS: 2, 4, 6, 8 and 10 depict the corresponding
encoded full-length KUZ proteins. Methods used to isolate
additional members of the kuz family are described below and in the
Examples.
[0013] Preferred translates/deletion mutants comprise at least a
10, preferably at least a 15, more preferably at least a 20 residue
domain of at least one of SEQ ID NOS:2, 4, 6, 8 and 10.
[0014] In particular, KUZ derivatives can be made by altering KUZ
sequences by substitutions, additions or deletions that provide for
functionally equivalent molecules. Due to the a degeneracy of
nucleotide coding sequences, other DNA sequences which encode
substantially the same amino acid sequence as a kuz gene may be
used in the practice of the present invention. These include but
are not limited to nucleotide sequences comprising all or portions
of kuz genes which are altered by the substitution of different
codons that encode a functionally equivalent amino acid residue
within the sequence, thus producing a silent change. Likewise, the
KUZ derivatives of the invention include, but are not limited to,
those containing, as a primary amino acid sequence, all or part of
the amino acid sequence of a KUZ protein including altered
sequences in which functionally equivalent amino acid residues are
substituted for residues within the sequence resulting in a silent
change. For example, one or more amino acid residues within the
sequence can be substituted by another amino acid of a similar
polarity which acts as a functional equivalent, resulting in a
silent alteration. Conservative substitutes for an amino acid
within the sequence may be selected from other members of the class
to which the amino acid belongs. For example, the nonpolar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine proline, phenylalanine, tryptophan and methionine. The polar
neutral amino acids include glycine, serine, threonine, cysteine,
tyrosine, asparagine and glutamine. The positively charged (basic)
amino acids include arginine, lysine and histidine. The negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid.
[0015] In a specific embodiment of the invention, proteins
consisting of or comprising a fragment of a KUZ protein consisting
of at least 10 (continuous) amino acids of the KUZ protein is
provided. In other embodiments, the fragment consists of at least
15 or 20 or 50 amino acids of the KUZ protein. In specific
embodiments, such fragments are not larger than 35, 100 or 200
amino acids. Derivatives or analogs of KUZ include but are not
limited to those peptides which are substantially homologous to a
KUZ protein or fragments thereof. (e.g., at least 30%, 50%, 70%, or
90% identity over an amino acid sequence of identical size-- e.g.,
comprising a domain) or whose encoding nucleic acid is capable of
hybridizing to a coding KUZ sequence.
[0016] The subject domains provide KUZ domain specific activity or
function, such as KUZ- specific protease or protease inhibitory
activity, disintegrin or disintegrin inhibitory aci:ivity,
ligand/antibody binding or binding inhibitory, immunogenicity,
etc.; see, e.g. domains identified in FIG. 1A-C. Preferred domains
cleave a NOTCH protein. KUZ-specific activity or function may be
determined by convenient in vitro, cell-based, or in vivo assays:
e.g. in vitro binding assays, cell culture assays, in animals (e.g.
gene therapy, transgenics, etc.), etc. Binding assays encompass any
assay where the molecular interaction of an KUZ polypeptide with a
binding target is evaluated. The binding target may be a natural
intracellular binding target such as an KUZ substrate, a KUZ
regulating protein or other regulator that directly modulates KUZ
activity or its localization; or non-natural binding target such a
specific immune protein such as an antibody, or an KUZ specific
agent such as those identified in screening assays such as
described below. KUZ-binding specificity may assayed by protease
activity or binding equilibrium constants (usually at least about
107M-, preferably at least about 108 M-, more preferably at least
about 109 M-), by the ability of the subject polypeptide to
function as negative mutants in KUZ-expressing cells, to elicit KUZ
specific antibody in a heterologous host (e.g a rodent or rabbit),
etc. The KUZ binding specificity of preferred KUZ polypeptides
necessarily distinguishes that of the bovine protein of Howard, L.,
et al. (1996). Biochem. J. 317, 45-50.
[0017] The claimed KUZ polypeptides are isolated or pure: an
"isolated" polypeptide is unaccompanied by at least some of the
material with which it is associated in its natural state,
preferably constituting at least about 0.5%, and more preferably at
least about 5% by weight of the total polypeptide in a given sample
and a pure polypeptide constitutes at least about 90%, and
preferably at least about 99% by weight of the total polypeptide in
a given sample. The KUZ polypeptides and polypeptide domains may be
synthesized, produced by recombinant technology, or purified from
mammalian, preferably human cells. A wide variety of molecular and
biochemical methods are available for biochemical synthesis,
molecular expression and purification of the subject compositions,
see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al.
Cold Spring Harbor Laboratory), Current Protocols in Molecular
Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-
Interscience, NY) or that are otherwise known in the art. Material
and methods for the expression of heterologous recombinant proteins
in bacterial cells (e.g. E. coli), yeast (e.g. S. Cerevisiae),
animal cells (e.g. CHO, 3T3, BHK, baculovirus-compatible insect
cells, etc.). The KUZ polypeptides and/or domains thereof may be
provided uncomplexed with other protein, complexed in a wide
variety of non-covalent associations and binding complexes,
complexed covalently with other KUZ or non-KUZ peptide sequences
(homo or hetero- chimeric proteins), etc.
[0018] The invention provides binding agents specific to the
claimed KUZ polypeptides, including substrates, agonists,
antagonists, natural intracellular binding targets, etc., methods
of identifying and making such agents, and their use in diagnosis,
therapy and pharmaceutical development. For example, specific
binding agents are useful in a variety of diagnostic and
therapeutic applications, especially where disease or disease
prognosis is associated with improper utilization of a pathway
involving the subject proteins. Novel KUZ-specific binding agents
include KUZ-specific receptors, such as somatically recombined
polypcptide receptors like specific antibodies or T-cell antigen
receptors (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory
Manual, Cold Spring Harbor Laboratory) and other natural
intracellular binding agents identified with assays such as one-,
two- and three-hybrid screens, non-natural intracellular binding
agents identified in screens of chemical libraries such as
described below, etc. Agents of particular interest modulate KUZ
function, e.g. KUZ- dependent proteolytic processing. For example,
a wide variety of inhibitors of KUZ Notch protease activity may be
used to regulate signal transduction involving Notch.
[0019] Metalloprotease and disintegrin inhibitors and methods for
designing such inhibitors are well known in the art, e.g.
Matrisian, L. TIG, 6:(1990), Hooper, N. FEBS Let. 354:1-6 (1994),
Haas et al., Cur. Op. Cell Bio. 6:656-662 (1994), etc. Exemplary
inhibitors include known classes of metalloprotease inhibitors,
KUZ-derived peptide inhibitors, esp. dominant negative deletion
mutants, etc. KUZ specificity and activity are readily quantified
in high throughput protease assays using panels of proteases.
[0020] Accordingly, the invention provides methods for modulating
signal transduction involving Notch in a cell comprising the step
of modulating KUZ protease activity, e.g. by contacting the cell
with a protease inhibitor. The cell may reside in culture or in
situ, i.e. within the natural host. For use in methods applied to
cells in situ, the compositions frequently further comprise a
physiologically acceptable excipient and/or other pharmaceutically
active agent to form pharmaceutically acceptable compositions.
Hence, the invention provides administratively convenient
formulations of the compositions including dosage units which may
be incorporated into a variety of containers. The subject methods
of administration generally involve contacting the cell with or
administering to the host an effective amount of the subject
compounds or pharmaceutically acceptable compositions.
[0021] The compositions and compounds of the invention and the
pharmaceutically acceptable salts thereof can be administered to a
host in any effective way such as via oral, parenteral or topical
routes. Preferred inhibitors are orally active in mammalian
hosts.
[0022] In one embodiment, the invention provides the subject
compounds combined with a pharmaceutically acceptable excipient
such as sterile saline or other medium, gelatin, an oil, etc. to
form pharmaceutically acceptable compositions. The compositions
and/or compounds may be administered alone or in combination with
any convenient carrier, diluent, etc. and such administration may
be provided in single or multiple dosages. Useful carriers include
solid, semi-solid or liquid media including water and non-toxic
organic solvents. In another embodiment, the invention provides the
subject compounds in the form of a pro-drug, ,which can be
metabolically converted to the subject compound by the recipient
host. A wide variety of pro-drug formulations are known in the art.
The compositions may be provided in any convenient form including
tablets, capsules, lozenges, troches, hard candies, powders,
;sprays, creams, suppositories, etc. As such the compositions, in
pharmaceutically acceptable dosage units or in bulk, may be
incorporated into a wide variety of containers. For example, dosage
units may be included in a variety of containers including
capsules, pills, etc.
[0023] The compositions may be advantageously combined and/or used
in combination with other therapeutic or prophylactic agents,
different from the subject compounds. In many instances,
administration in conjunction with the subject compositions
enhances the efficacy of such agents, see e.g. Goodman &
Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., 1996,
McGraw-Hill. For diagnostic uses, the inhibitors or other KUZ
binding agents are frequently labeled, such as with fluorescent,
radioactive, chemiluminescent, or other easily detectable
molecules, either conjugated directly to the binding agent or
conjugated to a probe specific for the binding agent.
[0024] According to the invention, a KUZ protein, its fragments or
other derivatives, or analogs thereof, may be used as an immunogen
to generate antibodies which recognize such an immunogen. Such
antibodies include but are not limited to polyclonal, monoclonal,
chimeric, single chain, Fab fragments, and an Fab expression
library. In a specific embodiment, antibodies to human KUZ are
produced. In another embodiment, antibodi es to the extracellular
domain of KUZ are produced. In another embodiment, antibodies to
the intracellular domain of KUZ are produced.
[0025] Various procedures known in the art may be used for the
production of polyclonal antibodies to a KUZ protein or derivative
or analog. In a particular embodiment, rabbit polyclonal antibodies
to an epitope of the KUZ protein encoded by a sequence selected
from SEQ ID NOS: 1, 3, 5, 7 or 9 or a subsequence thereof, can be
obtained. For the production of antibody, varioius host animals can
be immunized by injection with the native KUZ protein, or a
synthetic version, or derivative (e.g., fragment) thereof,
including but not limited to rabbits, mice, rats, etc. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and including but not limited to
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium
parvum.
[0026] For preparation of monoclonal antibodies directed toward a
KUZ protein sequence or analog thereof, any technique which
provides for the production of antibody molecules by continuous
cell lines in culture may be used. For example, the hybridoma
technique originally developed by Kohler and Milstein (1975, Nature
256: 495-497), as well as the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72),
and the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al., 1985, in Monoclonal antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96)>. In an additional
embodiment of the invention, monoclonal antibodies can be produced
in germ-free animals utilizing recent technology (PCT/US90/02545).
According to the invention, human antibodies may be used and can be
obtained by using human hybridomas (Cote et al., [983, Proc. Natl.
Acad. Sci. U.S.A. 80: 2026-2030) or by transforming human B cells
with EBV virus in vitro (Cole et al., 1985, in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact,
according to the invention, techniques developed for the production
of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature
312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing
the genes from a mouse antibody molecule specific for KUZ together
with genes from a human antibody molecule of appropriate biological
acitvity can be used; such antibodies are within the scope of this
invention.
[0027] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778) can
be adapted to produce KUZ-specific single chain antibodies. An
additional embodiment of the invention utilizes the techniques
described for the construction of Fab expression libraries (Huse et
al., 1989, Science 246:1275-1281) to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity for KUZ proteins, derivatives, or analogs.
[0028] Antibody fragments which contain the idiotype of the
molecule can be generated by known techniques. For example, such
fragments include but are not limited to: the F(ab).sub.2 fragment
which can be produced by pepsin digestion of the antibody molecule;
the Fab fragments which can be generated by reducing the disulfide
bridges of the F(ab).sub.2 fragment, and the Fab fragments which
can be generated by treating the antibody molecule with papain and
a reducing agent. In the production of antibodies, screening for
the desired antibody can be accomplished by techniques known in the
art e.g ELISA (enzyme-linked immunosorbent assay). For example, to
select antibodies which recognize a specific domain of a KUZ
protein, one may assay generated hybridomas for a product which
binds to a KUZ fragment containing such domain. For selection of an
antibody immunospecific to human KUZ., one can select on the basis
of positive binding to human KUZ and a lack of binding to a KIJZ of
another species. The foregoing antibodies can be used in methods
known in the art relating to the localization and activity of the
protein sequences of the invention, e.g., for imagi,ng these
proteins, measuring levels thereof in appropriate physiological
samples, in diagnostic methods, etc. Antibodies specific to a
domain of a KUZ protein are also provided. In a specific
embodiment, antibodies which bind to a Notch-binding fragment of
KUZ are provided.
[0029] The amino acid sequences of the disclosed KUZ polypeptides
are used to back-. translate KUZ polypeptide-encoding nucleic acids
optimized for selected expression systems (Holler et al. (1993)
Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used
to agenerate degenerate oligonucleotide primers and probes for use
in the isolation of natural KUZ-encoding nucleic acid sequences
("GCG" software, Genetics Computer Group, Inc, Madison WI).
KUZ-encoding nucleic acids used in KUZ-expression vectors and
incorporated into recombinant host cells, e.g. for expression and
screening, transgenic animals, e.g. for functional studies such as
the efficacy of candidate drugs for disease associated with
KUZ-modulated cell function, etc.
[0030] The invention also provides nucleic acid hybridization
probes and replication / amplification primers having a KUZ cDNA
specific sequence comprising SEQ ID NO: 1, 3, 5, 7 or 9, and
sufficient to effect specific hybridization thereto (i.e.
specifically hybridize with SEQ ID NO: 1, 3, 5, 7 or 9,
respectively, in the presence of an embryonic cDNA library from the
corresponding species, and preferably in the presence of BMP cDNA
as described by Howard and Glynn (1995). Such primers or probes are
at least 12, preferably at least 24, more preferably at least 36
and most preferably at least 96 bases in length. Demonstrating
specific hybridization generally requires stringent conditions,
i.e. those that (1) employ low ionic strength and high temperature
for washing, for example, 0.015 M NaCl/0.0015 M sodium titrate/0.1%
SDS at 50.degree. C., or (2) employ during hybridization a
denaturing agent such as formamide, for example, 50% (vol/vol)
formamide with 0.1% bovine serum albumi n/0.1% Ficoll/0. 1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM NaCl, 75 mM sodium citrate at 42.degree. C. Another example
is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 (g/ml), 0.10% SDS, and 10% dextran sulfate at 42.degree.
C., with washes at 42.degree. C. in 0.2.times.SSC and 0.1% SDS. KUZ
nucleic acids can also be distinguished using alignment algorithms,
such as BLASTX (Altschul et a. (1990) Basic Local Alignment Search
Tool, J Mol Biol 215, 403-410).
[0031] The subject nucleic acids are of synthetic/non-natural
sequences and/or are isolated, i.e. unaccompanied by at least some
of the material with which it is associated in its natural state,
preferably constituting at least about 0.5%, preferably at least
about 5% by weight of total nucleic acid present in a given
fraction, and usually recombinant, meaning they comprise a
non-natural sequence or a natural sequence joined to nucleotide(s)
other tham that which it is joined to on a natural chromosome.
Recombinant nucleic acids comprising the nucleotide sequence of SEQ
ID NO: 1, 3, 5, 7 or 9, or the subject fragments thereof, contain
such sequence or fragment at a terminus, immediately flanked by
(i.e. contiguous with) a sequence other than that which it is
joined to on a natural chromosome, or flanked by 2L native flanking
region fewer than 10 kb, preferably fewer than 2 kb, which is at a
terminus or is immediately flanked by a sequence other than that
which it is joined to on a natural chromosome. While the nucleic
acids are usually RNA or DNA, it is often advantageous to use
nucleic acids comprising other bases or nucleotide analogs to
provide modified stability, etc.
[0032] The subject nucleic acids find a wide variety of
applications including use as translatable transcripts,
knock-in/out vectors, hybridization probes, PCR primers, diagnostic
nucleic acids, etc.; use in detecting the presence of KUZ genes and
gene transcripts and in detecting or amplifying nucleic acids
encoding additional KUZ homologs and structural analogs. In
diagnosis, KUZ hybridization probes find use in identifying
wild-type and mutant KUZ alleles in clinical and laboratory
samples. Mutant alleles are used to generate allele- specific
oligonucleotide (ASO) probes for high-throughput clinical
diagnoses. In therapy, therapeutic KUZ nucleic acids are used to
modulate cellular expression or intracellular concentration or
availability of active KUZ.
[0033] The invention provides efficient methods of identifying
agents, compounds or lead compounds for agents active at the level
of a KUZ modulatable cellular function. Generally, these screening
methods involve assaying for compounds which modulate KUZ
interaction with a natural KUZ binding target such as a Notch
protein, etc. A wide variety of assays for binding agents are
provided including labeled in vitro protein-protein binding assays
including protease assays, immunoassays, cell based assays, etc.
The methods are amenable to automated, cost-effective high
throughput screening of chemical libraries for lead compounds.
Identified reagents find use in the pharmaceutical industries for
animal and human trials; for example, the reagents may be
derivatized and rescreened in in vitro and in vivo assays to
optimize activity and minimize toxicity for pharmaceutical
development.
[0034] Exemplary in vitro binding assays employ a mixture of
components including an KUZ polypeptide, which may be part of a
fusion product with another peptide or polypeptide, e.g. a tag for
detection or anchoring, etc. The assay mixtures comprise a natural
intracellular KUZ binding target. In a particular embodiment, the
binding target is a Notch protein-derived substrate of KUZ protease
activity. Such substrates comprise a specifically KUZ-cleavable
peptide bond and at least 5, preferably at least 10, and more
preferably at least 20 naturally occurring immediately flanking
residues on each side. While native full-length binding targets may
be used, it is frequently preferred to use portions (e.g. peptides)
thereof so long as the portion provides binding affinity and
avidity to the subject KUZ polypeptide conveniently measurable in
the assay. The assay mixture also comprises a candidate
pharmacological agent. Candidate agents encompass numerous chemical
classes, though typically they are organic compounds; preferably
small organic compounds and are obtained from a wide variety of
sources including libraries of synthetic or natural compounds. A
variety of other reagents may also be included in the mixture.
These include reagents like ATP or ATF analogs (for protease
assays), salts, buffers, neutral proteins, e.g. albumin,
detergents, non- specific protease inhibitors, nuclease inhibitors,
antimicrobial agents, etc. may be used.
[0035] The resultant mixture is incubated under conditions whereby,
but for the presence of the candidate pharmacological agent, the
KUZ polypeptide specifically binds the cellular binding target,
portion or analog with a reference binding affinity. The mixture
components can be added in any order that provides for the
requisite bindings and incubations may be performed at any
temperature which facilitates optimal binding. Incubation periods
are likewise selected for optimal binding but also minimized to
facilitate rapid, high-throughput screening.
[0036] After incubation, the agent-biased binding between the KUZ
polypeptide and one or more binding targets is detected by any
convenient way. For KUZ protease assays, `binding` is generally
detected by the generation of a KUZ substrate cleavage product. In
this embodiment, protease activity may quantified by the apparent
transfer a label from the substrate to the nascent smaller cleavage
product, where the label may provide for direct detection as
radioactivity, luminescence, optical or electron density, etc. or
indirect detection such as an epitope tag, etc. A variety of
methods may be used to detect the label depending on the nature of
the label and other assay components, e.g. through optical or
electron density, radiative emissions, nonradiative energy
transfers, etc. or indirectly detected with antibody conjugates,
etc.
[0037] A difference in the binding affinity of the KUZ polypeptide
to the target in the absence of the agent as compared with the
binding affinity in the presence of the agent indicates that the
agent modulates the binding of the KUZ polypeptide to the KUZ
binding target. Analogously, in cell-based assays described below,
a difference in KUZ-depenclent modulation of signal transduction in
the presence and absence of an agent indicates the agent modulates
KUZ fulnction. A difference, as used herein, is statistically
significant and preferably represents at least a 50%, more
preferably at least a 90% difference.
[0038] Altered Drosophila hosts in which the kuz gene is
over-expressed, under-expressed, mis-expressed or expressed as a
variant are used to identify compounds that are antagonist or
agonists of the KUZ polypeptide as well as to identify genes that
encode products that interact with the KUZ polypeptide using art
known methods (Xu et al., Genes and Devel., p464-475 (1990), Simon
et al., Cell, 67:701-716 (1991) and Fortini et al., Cell,
79:273.282 (1994)).
[0039] Agents that modulate the interactions of the KUZ polypeptide
with its ligands/natural binding targets can be used to modulate
biological processes associated KUZ function, e.g. by contacting a
cell comprising a KUZ polypeptide (e.g. administering to a subject
comprising such a cell) with such an agent. Biological processes
mediated by KUZ polypeptides include a wide variety of cellular
events which are mediated when a KUZ polypeptide binds a ligand
e.g. cell differentiation, cell development and neuronal
partitioning. The agents are also used to modulate processes
effected by KUZ substrates; for example, Notch, an art known
peptide involved in neurogenesis is over-expressed in some forms of
leukemia (Ellison et al., Cell, 30 66:649-661 (1991)).
[0040] The present invention further provides methods for
identifying cells involved in KUZ polypeptide-mediated activity,
e.g. by determining whether the KUZ polypeptide, or a kuz ligand,
is expressed in a cell. Such methods are useful in identifying
cells and events involved in neurogenesis. In one example, an
extract of cells is prepared and assayed by of a variety of
immunological and nucleic acid techniques to determine whether the
KUZ polypeptide is expressed. The presence of the KUZ polypeptide
provides a measurement of the participation or degree of
neurogenesis of a cell.
[0041] The invention provides a wide variety of methods and
compositions for evaluating modulators of the KUZ signaling
pathways. For example, the invention provides transgenic non-human
animals such as flies (e.g. Drosophila), worms (e.g. C. elegans),
mice, etc. having at least one structurally and functionally
disrupted KUZ allele. In particular embodiments, the animals
comprise a transgene within a KUZ allele locus, encoding a
selectable marker and displacing at least one exon of the KUZ
allele. More particularly, the transgene may comprise 3' and 5'
regions with sufficient complementarity to the natural KUZ allele
ait the locus to effect homologous recombination of the transgene
with the KUZ allele. Such animals provide useful models for
determining the effect of candidate drugs on a host deficient in
KUZ function.
[0042] As describe above, the invention provides a wide variety of
methods for making and using the subject compositions. As
additional examples, the invention provides methods for (Yf,
determining the effect of a candidate drug on a host deficient in
KUZ function, such as:
[0043] contacting a transgenic animal having at least one disrupted
KUZ allele with a candidate drug; and, detecting the presence or
absence of a physiological change in the animal in response to the
contacting step. The invention also provides methods of evaluating
the side effects of a KUZ function inhibitor, such as: contacting a
transgenic animal having at least one disrupted KUZ allele with a
candidate drug; detecting the presence or absence of a
physiological change in the animal in response to the contacting
step, wherein the presence of a physiological change indicates the
inhibitor has side effects beyond KUZ function inhibition.
[0044] Without further description, one of ordinary skill in the
art can, using the preceding description and the following
illustrative examples, make and utilize the compounds of the
present invention and practice the claimed methods. The following
working examples therefore, specifically point out preferred
embodiments of the present invention, and are not to be construed
as limiting in any way the remainder of the disclosure. Other
generic configurations will be apparent to one skilled in the art.
All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
EXAMPLES
Example 1: Identification of a Drosophila KUZ polypeptide/gene
[0045] Genes involved in lateral inhibition were screened using
FLP/FRT chromosomes to produce mutant clones in mosaic animals (T.
Xu and G.M. Rubin, Development 117:12273 (1993); T. Xu and S.
Harrison Methods in Cell Biology 44:655 (1994)) and to isolate
several alleles of a gene family designated herein as kuzbanian
(kuz). The kuz locus is defined by a single complementation group
which maps to chromosomal location 34C4,5, and corresponds to the
1(2)34 Da group (A.C. Spradling et al., PNAS 92:10824 (1995). Most
of the kuz phenotypic analysis was performed using the null allele
kuze29-4. Kuze29-4 is an excision allele deleting approximately 2.4
kb at the 5' end of the kuz gene, including DNTA in the promoter
region and the first and second exons. Four P[lacZ; w+] insertions
1(2)kl 1804, 1(2)kO1403, 1(2)kO7601 and 1(2)kl4701 are hypomorphic
kuz alleles. These insert either in the first kuz exon or in the
first intron. Precise excision of these P insertions reverts the
associated kuz phenotype. Kuzl is the original kuz allele caused by
an insertion of 4.3 kb of DNA in or near the first exon. Seventeen
additional X-ray induced kuz alleles were isolated in the FLP/FRT
mosaic screen.
[0046] A 10 kb fragment of DNA from the region deleted in allele
kuze29-4 was used to) screen a Drosophila total imaginal disc cDNA
library. A group of two overlapping 1.2 kb cDNAs mapping to this
region was recovered; a fiull-length kuz cDNA, NB 1, was isolated
from an embryonic cDNA library using the small cDNA clones as
probes (Kuz cDNA Genbank accession number: U60591).
[0047] Scanning electron microscopy (SEM) and embryo staining and
adult eye sections were carried out following standard procedures
(A. Tomlinson and D.F. Ready, Dev. Biol. 123:264 (1987); T. Xu and
S. Artavanis-Tsakonas, Genetics 126, 665 (1990)). A scanning
electron micrograph (SEM) showing the multiple bristle phenotype in
an adult mosaic fly with homozygous kuz clones revealed that
aeveral macro- and microchaete positions have supernumerary
bristles whereas others are missing in the same area. SEMs showing
kuz clones in the eye revealed the regular array of ommatidia is
severely disrupted, that toward the center of the clone the density
of photoreceptors is abnormally low and none are successfully
organized into ommatidia, and that chimeric ommatidia at the clone
border contain a mixture of pigmented wild-type photoreceptor cells
and mutant, unpigmented photoreceptors. Confocal images of embryos
stained with the neuronal-specific anti-Elav antibody demonstrate a
requirement for maternal and zygotic kuz products. A kuz maternal
null embryo (generated using the ovoD mutation as described in T.B.
Chou and N. Perrimon, Genetics 131:643 (1992)) with one zygotic
copy of kuz revealed that a greater proportion of the embrvo
developed as neural tissue than in wild-type and a surface view of
a kuz null embryo with no maternal or zygotic kuz product showed
that most cells adopted a neural fate. A lower focal plane of this
same embryo showed that all cells around the periphery of the
embryo are neural cells. A cuticular preparation of a kuz maternal
null embryo with one zygotic copy of kuz showed a small patch of
cuticle develops on the dorsal side of the embryo; presumably the
remaining cells which failed to produce cuticle adopted a neural
fate, consistent with the previously phenotype. A cuticular
preparation of a kuz null embryo showed only a tiny dot of cuticle
developed. Most of these embryos show no cuticle at all.
[0048] Animals with kuz mutant clones exhibit clusters of sensory
bristles at positions in which single sensory bristles are normally
observed. Separate sockets are often seen with individual bristles,
and stimulation of mutant bristles in a reflex test elicits a leg
cleaning response, indicating that mutant clusters contain multiple
sensory bristles and not just multiple shafts (P. Vandervorst and
A. Ghysen, Nature 286:65 (1980)). This multiple bristle phenotype
is observed in clones mutant for several neurogenic genes such as
Notch (N) and shaggy (sgg, also known as zeste-white 3), and is
indicative of a failure of lateral inhibition during the
development of the peripheral nervous system (S.
Artavanis-Tsakonas, et al., Trends in Genetics 7:403 (1991); J.S.
Campos-Ortega (1993); Jan, Y.N. and Jan, L.Y., id., pp. 1207-1244;
Romani, S. et al., Genes Dev. 3:997 (1989); Artavanis-Tsakonas, S.
et al., Science 268:225 (1995); Heitzler, P. and Simpson, P.
(1991). Cell 64, 1083-1092).
[0049] Unlike the N phenotype, kuz clones do not produce ectopic
bristles, indicating kuz is not required for correct spacing
between proneural clusters. Mutant clones in the adult eye severely
disrupted the regular array of ommatidia. Thin sections through
such a mosaic eye reveal that mutant photoreceptors are not
organized correctly into ommatidia.
[0050] To determine whether the KUZ polypeptide is required for the
development of the central nervous system (CNS), embryos lacking
any maternally derived KUZ polypeptide and containing one or no
zygotic copies of the kuz gene were produced. The embryos were
examined by staining with neuronal-specific antibodies to the Elav
protein (Bier, E. et al., Science 240:913 (1988); Robinow, S. et
al., J Neurobiol. 22, 443 (1991)). Maternal mill embryos with one
copy of zygotic kuz gene showed hyperplasia and disorganization of
the CNS on the ventral side of the embryos, which is a phenotype
similar to the neurogenic phenotype of N mutant embryos (Lehmann,
R. et al., Roux's Arch Dev. Biol. 192:62 (1983)). However, embryos
lacking all maternal and zygotic KUZ polypeptide have a much more
severe neurogenic phenotype. Hypertrophy of the nervous system is
not restricted to thle ventral region, but the embryos stained
throughout with anti-Elav, demonstrating that many more cells in
the embryo had developed as neural cells. Such a severe neuralizing
phenotype is similar to that of sgg null embryos (Bourouis, M. et
aL, Nature 341:442 (1989)). Cuticular preparation of embryos
correlated well with the antibody results: Maternal-null embryos
with one copy of the kuz gene produced a small patch of cuticle on
the dorsal side, consistent with the observation that many of the
ventral cells had adopted a neural fate at the expense of
epidermis. Embryos with no KUZ polypeptide produced little or no
cuticle, as would be expected if most cells had become neural,
leaving few epidermal cells to secrete cuticle:.
[0051] Further analyses on the development of adult sensory
bristles were performed to determine a specific role for the KUZ
polypeptide in lateral inhibition. The yellow (y) and crinkle (ck)
marker mutations were used to mark kuz- clones in the adult
cuticle. This allows one to determine the genotype of individual
cells and thus to examine the autonomy of the kuz mutant phenotype.
Such analysis can distinguish between sending and receiving roles
for a gene involved in the lateral inhibition process (Heitzler, P.
et al., Cell 64:1083 (1991 )).
[0052] A role for the KUZ polypeptide in lateral inhibition is
suggested by the observation that all sensory bristles in a mutant
cluster are kuz-; no wild-type bristles are ever present in a
cluster. SEM of kuz- clones (each kuz- cell is also ck- and y-)
revealed that the ck- mutation results in extra trichomes in the
epidermal cell and in blunted shafts of sensory bristles; these
morphological changes allow the border between mutant and wild-type
cells to be precisely determined. A marked absence of all micro-
and macrochaetes is observed in the interior of the clone, as no
shafts, sockets, or neurons (naked cells) are seen. Kuz- mutant
cells at normal bristle positions do form bristles at clone borders
where they are in contact withL wild-type cells. A high-
magnification view of one of the multiple macrochaete clusters at a
clone border revealed that every bristle in this and other clusters
is always ck- and y-, demonstrating that all bristles in a cluster
are kuz-. No wild-type bristles are observed in multiple bristle
clusters. Marked kuz- clones were generated in y- w- hsFLP];
kuse29-4ck-P[FRT}40A/P[y+] P[w+]P[FRT]40A first instar larvae
following protocols described in T. Xu and G.M. Rubin, Development
117:1223 (1993) and T. Xu and S. Harrison Methods in Cell Biology
44:655 (1994).
[0053] Mosaic analysis for kuz- clones in the adult cuticle
indicates two distinct functions for the kuz protein. First, the
failure of lateral inhibition, evidenced by the formation of extra
bristles, only occurs in kuz- mutant cells. This cell-autonomous
mutant phenotype indicates that during normal development, the kuz
protein is required in cells to receive an inhibitory signal. kuz-
cells at normal bristle-forming positions become bristles only when
they are in contact with wild-type cells, indicating that in
wild-type animals, the KUZ polypeptide may act as a positive signal
or is involved in sending a positive signal for the development cf
the bristle. Thus, there is a cell-autonomous requirement for kuz
in order for cells to be inhibited from adopting a neural precursor
fate. We conclude that the KUZ polypeptide is required in cells to
receive an inhibitory signal from the emerging neural cell. Cells
in the proneural cluster with wild-type KUZ polypeptide flnction
receive the inhibitory signal and are forced to become epidermal,
whereas kuz- cells cannot be inhibited and develop as neural
precursor cells. A second distinct role for the KUZ polypeptide was
revealed by the same mosaic, analyses. All mutant bristle clusters
were produced at clone borders, where mutant cells contact
wild-type cells. No bristles were ever produced in clone interiors,
either singly or in clusters. Large kuz- clones therefore cause
bare patches devoid of bristles containing only hair-secreting
epidermal cells. This phenotype indicates there is a non
cell-autonomou.s requirement for the KUZ polypeptide in bristle
development. Hence, Kuz participates in both neural-promoting and
-inhibiting processes during formation of the adult epidermis.
[0054] To reveal the molecular basis of the KUZ polypeptide
functions, a kuz gene was cloned and a full-length cDNA was
obtained. The kuz cDNA contained an open reading frame that encodes
al ,239 amino acid membrane-spanning protein of the
metalloprotease-disintegrin family known as the ADAM family
(members of the ADAM family contain "A Disintegrin And
Metalloprotease Domain". The KUZ metalloprotease domain also
contains a conserved zinc-binding site (Jiang, W. and Bond, J. S.
(1992). ],EBS Letters 312, 110-114). Like other disintegrins KUZ
has a characteristic spacing of cysileine residues that is required
for their direct binding to receptors (Niewiarowski, S. et al.,
Seminars in Hematology 31:289 (1994)). These cysteines are
conserved in the KUZ polypeptide along with many additional
residues that are shared by other disintegrin domains. In this
family, many proteins with a multi-domain structure are
proteolytically processed to produce multiple peptides with
different functions (Blobel, C.P. et al., J Cell Biol. 11 :69
(1990); Neeper, M.P. et al, Nucleic Acids Res. 18:4255 (1990); Au,
L.C., et al., Biochem. Biophys. Res. Commun. 181:585 (1991)). The
metalloprotease and disintegrin domains of kuz may be cleaved from
the full-length precursor to produce both soluble and
membrane-bound forms of these domains. Such proteolytic products of
the KUZ polypeptide may be used to carry out the different KUZ
polypeptide functions.
Example 2: Identification of two human and one mouse KUZ
polypeptides/genes.
[0055] The nucleic acid sequence of the Drosophila kuz gene was
used to generate PCR primers for amplifying kuz encoding nucleic
acid molecules from organisms other than Drosophila. A .9kb cDNA
fragment was amplified from a human fetal brain cDNA library
(Clonetech, Stratagene) using PCR primers. This fragment was cloned
and was used as a probe to screen the human fetal brain cDNA
library (Clonetech, Stratagene). A clone containing a 3.5kb insert
was obtained (SEQ ID NO:3). The cloned contained a full length
encoding sequence that encodes a protein of 749 amino acids. Three
additional clones were obtained that showed variant restriction
digestion patterns. Sequence analysis of these clones identified a
second form of the human KUZ polypeptide. This second form of the
KUZ polypeptide encodes a protein of 330 amino acids in length (SEQ
ID NO:6). A fragment of the human kuz encoding sequence was used to
probe a mouse fetal brain cDNA library. One of four isolated clones
was sequenced and contained a 4kb insert representing a murine KUZ
cDNA (SEQ ID NO:7).
[0056] Northern blots run using RNA isolated from various mouse and
human tissues revealed expression in fetal and adult tissues.
Hybridization of the blots with probes specific to each of the
human forms confirmed that each of the transcripts was unique to
one of the two forms, indicating that the two identified mRNA
transcripts represent each of the two human forms respectively. The
variable pattern of expression seen on the adult and fet-al
Northern blots indicates a developmental role of the KUZ
polypeptides: the short form being predominant in adult tissues
while the full length form is predominant in fetal tissues and
adult brain. All regions of the adult brain expressed both
forms.
Example 3: KUZBANIAN controls proteolvtic processing of NOTCH and
mediates lateral inhibition during Drosophila and vertebrate
neurogenesis.
[0057] To investigate how the different domains of KUZ contribute
to its biological functions, full length and various N- and C-
terminal truncations of KUZ were generated (e.g. constructs 1-4 and
7, FIG. 1B) and expressed under the pGMR vector (Hay, B. A., Wolff,
T. and Rubin, G. M. (1994). Development 120, 2121-2129) in the
developing retina of Drosophila. One of these exemplary truncations
(7), which is missing the protease domain, resulted in a dominant
rough eye phenotype. We expressed KUZ truncations using the pDMR
vector which contains the decapentaplegic (dpp) disc specific
enhancer element (see experimental procedures) that drives gene
expression in several tissues including parts of the notum and the
wing blade, two tissues that are known to be affected in kuz mutant
clones. Expression of construct 7 under pDMR resulted in
supernumerary bristles on the notuns and notches on the wing
blades. These phenotypes resemble those seen in somatic clones
homozygous for kuz loss-of-function mutations, indicating that this
construct functions in a dominant negative manner by interfering
with endogenous kuz activity. We also observed that the mutant
phenotypes resulting from this construct are dominantly enhanced by
removing one copy of the endogenous kuz gene; that is, the
phenotypes of kuzl+individuals carrying this construct are more
severe than those of +/+individuals. Conversely, additional
wildtype KUZ protein from a transgene expressing full length KUZ
suppresses these phenotypes. We refer to the particular dominant
negative of construct 7 hereafter as KUZDN (KUZ dominant
negative).
[0058] To directly address the functional relevance of the protease
domain, we introduced into full length KUZ a point mutation (E606
to A) in the putative zinc binding site (FIG. 1 A) of the protease
domain. This glutamate is thought to be a catalytic residue and is
absolutely conserved among all known metalloproteases (Jiang and
Bond, 1992). Thus, this point mutation should abolish protease
activity while having minimal impact on the other activities of
KUZ. Indeed, overexpression of KUZ.sup.E606A (construct 8 in FIG.
1B) gave similar, although somewhat weaker, dominant phenotypes to
those seen with KUZDN.
[0059] The notums of Drosophila adults carry two types of sensory
bristles, macrochae-tes and microchaetes. The sensory organ
precursor cells (SOPs) that generate the macrochaetes are selected
from pools of equivalent cells by lateral inhibition mostly during
the third instar larval stage, while the SOPs for the microchaetes
are singled out during the early pupae stage (Huang, F., et al.
(1991). Development 111, 1087-1095; Hartenstein, V. and Posakony,
J. W. (1989). Development 107, 389-405). N is required for this
process and removal of N function at larval and pupal stages
differentially affects these two types of bristles (Hartenstein, V.
and Posakony, J. W. (1990). Dev. Biol. 142, 13-30). If KUZ is
required for lateral inhibition, we would expect to generate
similar phenotypes by expressing KUZDN at these times. We generated
flies containing KUZDN under the control of the hsp70 promoter, and
applied one hour heat pulses at various times during larval and
pupal development. We observed that while heat pulses applied
during third instar larval stage resulted in supernumerary
macrochaetes only, heat pulses applied during early pupal stages
(0-13 hrs after uparitm formation (APF)) resulted in supernumerary
microchaetes only, similar to the phenotypes resulted from removing
N function at these times using a temperature sensitive N allele
(Hartenstein and Posakony, 1990). These time points match the
periods when SOPs for each bristle type are selected from pools of
equivalent cells (Huang et al., 199 1; Hartenstein and Posakony,
1989), indicating that KUZDN interferes with lateral inhibition
during the selection of SOPs.
[0060] Kuz mutant clones affect other tissues such as the eye. We
perturbed kuz functions by expressing KUZDN under the control of
the rough enhancer, which drives gene expression in all cells
within the morphogenetic furrow as well as transiently in R2, R5,
R3 and R4 posterior to the furrow (Heberlein, U., et al. (1994).
Mech. Dev. 48, 35-49). Flies carrying the rough/KUZDN transgene had
supernumerary photoreceptor cells in each ommatidium. Neuronal
differentiation in these transgenic flies was followed by staining
for ELAV, a. neuronal marker, in eye imaginal discs. Consistent
with the adult eye phenotype, we observed the recruitment of extra
neurons into each ommatidial cluster in the developing retina.
These experiments indicate that kuz finction is required to limit
the number of photoreceptor neurons recruited into each
ommatidium.
[0061] Besides its functions in determining neural fate, kuz is
also required for axonal extension at later stages of neural
development (Fambrough, D., et al. (1996). Proc. Natl. Acad. Sci.
USA 93, 13233-13238). We expressed KUZDN under the control of the
ELAV promoter using the GAL4-UAS system (Brand, A. H., and
Perrimon, N. (1993). Development 118, 401-415). The ELAV promoter
drives gene expression in maturing and mature neurons, but not
neuroblasts, thus allowing one to bypass the requirement for kuz in
neural fate determination. We observed that embryos expressing
KUZDN in developing neurons show major defects in axonal pathways,
such as disruption of longitudinal axonal tracts. In general, this
phenotype is similar to the that observed in zygotic kuz mutant
embryos (FambrougEh et al., 1996), indicating that KUZ provides a
proteolytic activity synthesized by axons and. required by them to
extend growth cones through the extracellular matrix.
[0062] Database searches revealed sequences representing putative
kuz orthologs in C. elegans, rat, bovine and human. The bovine
homolog was initially isolated as a proteolytic activity on myelin
basic protein in vitro (Howard et al., 1996). We isolated and
sequenced cDNAs representing a full-length mouse kuz homolog. This
mouse protein (MKUZ) is 45% identical in primary sequence with
Drosophila KUZ (DKUZ, FIG. 1), and 95% identical with the bovine
protein. Sequence similarity between MKUZ and DKUZ extends over the
whole coding region, except that MKUZ, like other vertebrate KUZ
homologs, has a much shorter intracellular domain. The
intracellular domain of MKUZ contains a stretch of 9 amino acid
residues (934-942) that are absolutely conserved with DKUZ. To
determine the functional importance of this sequence similarity, we
introduced into KUZDN mutations in several conserved residues in
this region (936TPSS939 to AAAA; construct 9 in Fig. lB) and found
these mutations dramatically reduced KUZDN activity.
[0063] Based on the structure of KUZDN described above, we
engineered a dominant negative form of MKUZ (MKUZDN, FIG. 1B)
missing the protease domain. When overexpressed in Drosophila using
the pDMR vector, MKUZDN resulted in dominant phenotypes resembling
those created by its Drosophila counterpart. To test directly the
involvement of MKUZ in vertebrate neurogenesis, we injected in
vitro transcribed mRNA encoding MKUZDN into Xenopus embryos.
Primary neurons in Xenopus are generated in precise and simple
patterns and can be identified by their expression of a neural
specific fr tubulin gene (N-tubulin). This assay has been used
previously to demonstrate a conserved role for certain neurogenic
genes in singling out primary neurons in Xenopus by lateral
inhibition (Chitnis, A., et al. (1995). Nature 375, 761-766). If a
kuz-like activity is required for the lateral inhibition process in
Xenopus, we would expect interference with this endogenous kuz
activity to result in the overproduction of primary neurons.
Indeed, injection of mRNA encoding MKUZDN resulted in extra
N-tubulin positive cells. Consistent with the notion that kuz acts
to limit the number of cells that differentiate as neurons from a
group of competent cells, these extra N-tubulin positive cells were
confined to domains of primary neurogellesis, and-were not observed
at ectopic positions.
[0064] To provide further evidence for an endogenous kuz activity
during primary neurogenesis in Xenopus, we examined the expression
pattern of a Xenopus kuz homolog (Xkuz). A cDNA fragment
representing a portion of Xkuz (FIG. 1) was isolated (see
experimental procedures) and used to generate RNA probes for in
situ hybridization under high stringency. Xkuz is expressed
uniformly in gastrulating and neural plate stage embryos, including
the domains of primary neurogenesis. In older embryos, xkuz
continues to be widely expressed, with an elevated level in neural
tissues. Thus, Xkuz is expressed at the appropriate time and place
for a potential role in primary neurogenesis in Xenopus. We sought
to determine the order of action of N and kuz by examining the
phenotype produced by combining a gain-of-function N mutant and a
loss-of-function kuz mutant. Expression of an activated form of
NOTCH (reviewed in Artavanis-Tsakonas et al., 19)5) under the heat
shock promoter (hs-N.sup.act) at early pupal stages (7-9 hours APF)
leads to the loss of microchaetes on the notum; the opposite
phenotype, extra microchaetes, is seen in loss-of-function kuz
mutant clones. We focused on microchaetes since the SOPs for these
bristles are generated more synchronously than those of the
macrochaetes (Huang et al., 1991; Hartenstein and Posakony, 1989)
and thus a single pulse of heatshock at 7-9 hrs APF results in the
reproducible loss of microchaetes on the notum in hs-N flies. If
kuz acts genetically downstream of N, then the combination of N and
kuz should display the kuz phenotype of extra microchaetes.
Conversely, if kuz acts genetically upstream of N, then the
combination of N and kuz should display the N phenotype of missing
microchaetes. We observed that the combination of N.sup.act and kuz
displayed the N.sup.act phenotype, indicateing that kuz acts.
genetically upstream of N. This result indicates KUZ acts upstream
of, or parallel with NOTCH in the same biochemical pathway.
[0065] We observed dosage sensitive genetic interactions between
kuz and N, indicating that the levels of activity of kuz and N are
tightly balanced. We took advantage of a weak dpp-KUZDN transgene
that resulted in an average of 3 posterior scutellar bristles
instead of the 2 seen in wildtype. While heterozygous N mutants
have normal number of posterior scul.ellar bristles, this genetic
background dramatically enhanced the phenotype resulting from the
weak dpp-KUZDN transgene such that an average of 5.2 bristles
(n=50) were observed. Furthermore, in flies that carry an
additional copy of N gene, the extra bristle phenotype resulting
from this KUZDN transgene is completely suppressed such that 2
bristles were observed. This intricate balance between their
activities indicates that kuz and N are closely linked in a common
biological process.
[0066] We examined if perturbation of KUZ function in Drosophila
Schneider 2 (S2) cell cultures would affect NOTCH processing. S2
cells do not express any endogenous NOTCH protein (Fehon et al.,
1990), but do express high levels of kuz mRNA. Upon transfection of
a full-length N construct, the monoclonal antibody C17.9C6, which
was raised against the intracellular domain of NOTCH, can detect
full length NOTCH (about 300 kd) and C- terminal fragments of about
100 kd (Fehon et al., 1990). We reasoned that if kuz is involved in
generating this 100 kd species in S2 cells, then expression of
KUZDN might interfere with this proteolytic event. Indeed,
expression of KUZDN nearly abolished the 100 kd species in S2
cells, while the 300 kd species was not greatly affected,
indicating that kuz is required for the NOTCH processing.
Consistent with our results in transgenic flies that overexpression
of full length KUZ did not perturb neurogenesis, transfection of a
full length KUZ construct did not affect NOTCH processing in S2
cells.
[0067] Next, we performed similar experiments in developing
imaginal discs. As described earlier, in transgenic flies
containing KUZDN under the control of the heatshock promoter, one
hour heatshock at the third instar larval stage resulted in extra
bristles on the notum. The same heatshock regime also resulted in
notches on the wing blade and extra photorec(ptors in the eye. We
followed the status of NOTCH processing in the wing and eye
imaginal discs after the induction of KUZDN in these animals. As in
transfected S2 cells, mAb C17.9C6 normally detects a 300 kd and a
100 kd NOTCH species in protein extracts of the third instar
imaginal discs. After the induction of KUZDN by one hour heatshock,
the I 00 kd species gradually disappears; by 4 hours after
induction, the 100 kd species is almost undetectable, while the 300
kd species has accumulated to a higher level. By 15 hrs after the
heatshock, the 100 kd species is restored to wildtype levels
presumably reflecting the decay of the KUZDN protein synthesized in
response to the heatshock. The correlation between the reduction of
the 100 kd species upon KUZDN expression and the resulting
neurogenic phenotypes in imaginal tissues indicates the functional
significance of the 100 kd NOTCH form detected in vivc.
[0068] Finally, we examined NOTCH processing in ktiz null mutant
embryos. Since kuz is known to have a maternal contribution
(supra), we generated germline clones to obtain embryos lacking all
KUZ function. We found that while mAb C17.9C6 detects a 300 kcd and
a 100 kd species in wildtype embryos, only the 300 kd species is
detected in kuz null embryos. This observation indicates that the
phenotypes we generated by expression of KUZDN are not due to
interference with genes other than kuz, such as other members of
the ADAM family, and that kuz is required for the proteolytic
processing of NOTCH (FIG. 2).
[0069] Our studies provide a general scheme for engineering
dominant negative forms of ADAM proteins applicable to other ADAM
genes. While all ADAMs possess a disintegrin- like and a
metalloprotease-like domain, some ADAMs lack a consensus active
site in the metalloprotease domain. These "protease dead" ADAMs
resemble dominant negative forms of KUZ described herein and can
function as endogenous inhibitors.
[0070] Experimental Procedures: Plasmid Constructs: We initially
used the pGMR vector (Hay et al., 1994) to express full length KUZ
and several N- and C- terminal deletion constructs in the eye.
These constructs include 1, 2, 3, 4 and 7. Upon identification
of-as a dominant negative form (KUZDN), we then used another
expression vector pDMR to express constructs 1, 4, 5, 6, 7, 8 and
9. The pDMR vector utilizes the dpp disc specific enhancer to drive
gene expression in multiple tissues including the wing and the
notum. pDMR was constructed by the following steps. First, the heat
shock responsive element in CasperhEs (Pirotta, V. ( 1988). In
Vectors: A Survey of Molecular Cloning Vectors and their Uses) was
removed to yield Casperhs-1. A 4.3 kb dpp disc specific enhancer
(Staehling-Hamptort, K., et al.(1994). Cell Growth Differ. 5,
585-593) was inserted upstream of the hsp7O basal promoter in
Casperhs- I to yield pDMR (dpp mediated reporter). Construct 7
(KUZDN) was also cloned into pUAST (Brand and Perrimon, 1993) and
pCasperhs to generate UAS/KUZDN and hs/KUZDN, respectively. A rough
enhancer element (Heberlein et al., 1994) was then inserted into
hs/KUZDN to generate rough/KUZDN. Constructs 1 (full length KUZ)
and 7 (KUZDN) were also cloned downstream of the metallothionein
promoter in pRMHa-3, a S2 cell expression vector (Bunch, T. A., et
al. (1988) Nucl. Acids Res. 16, 1043-1061). The nucleotide
coordinates of constructs I through 9 are as follows, using the
same numbering as in GenBank accession no. U60591. 1 and 8:
723-5630; 2: 723-3578; 3: 723-3462; 4: 723- 2757; 5: 1957-2757; 6:
1957-5630; 7 and 9: 2757-5630. Note that for all the N- terminal
deletion constructs, a DNA fragment (nucleotides 723-940)
containing the signal peptide was provided at the 5end. Site
directed mutagenesis was carried out using Stratagene's QuickChange
system.
[0071] MKUZDN was generated by an N- terminal truncation that
removes the pro and catalytic domains of MKUZ. The rest of MKUZ
(nucleotide 1483-2573) was ligated either to a DNA fragment
(723-940, according to nucleotide coordinates in U60591) containing
the signal peptide of Drosophila KUZ to generate MKUZDN-1 or to a
fragment (nucleotide 1- 248) containing the signal peptide of MKUZ
to generate MKUZDN-2. MKUZDN- I was subcloned into pDMR and pUAST
for overexpression in Drosophila, and MKUZDN-2 was subdloned into a
modified CS2+vector (Turner, D. L. and Weintraub, H. (1994). Genes
Dev. 8, 1434-1447.) for RNA injection in Xenopus embryos (see
below). Characterization of kuz Homologs from Mouse and Xenopus:
PCR primers corresponding to sequences of a rat gene similar to kuz
(GenBank accession: Z48444) were used to amplify a fragment from a
mouse brain cDNA library. PCR product was then used to screen
oligo(dT) and random primed cDNA libraries from the mouse PCC4 cell
line (Stratagene). Two overlapping cDNA, mkuz2 and mkuz3 were
characterized and sequenced, which together comprised the whole
coding region. mkuz 2 extends from nucleotide 430 to 2573 and mkuz3
extends from I to 1345.
[0072] Xenopus kuz was cloned by PCR using degenerate primers (XKI)
and (XK4) which correspond to Drosophila KUZ sequence HNFGSPHD and
GYCDVF, respectively. First strand cDNA from stage 18 Xenopus
embryos was used as template in a standard PCR reaction with an
annealing temperature of 50.degree. C. A PCR product of expected
size was purified and used as template for another PCR reaction
using a nested primer (XK3), corresponding to Drosophila KUZ
sequence EECDCG, and XK4. The PCR product was subcloned into
Bluescript and sequenced. Anti-sense RNA was used as a probe for
whole mount in situ hybridization of Xenopus embryos according to
standard procedures (Harland, R. (1991,. Meth. Cell Biol. 36,
685-695).
[0073] For RNA injections in Xenopus embryos, MKUZDN-2 was
synthesized in vitro using SP6 RNA polymerase from a CS2+vector.
Nuclear lacZ RNA was synthesized from plasmid pSP6nucB3Gal. 500 pg
of MKUZDN RNA, together with 100 pg of lacZ RNA was injecLed into
one blastomere of Xenopus embryos at 2-4 cell stage. lacZ RNA was
also injected alone as a control. Embryos were fixed at the neural
plate stage and stained with Red-Gal (Re search Organics, Inc.).
Embryos were then processed for in situ hybridization with a neural
spe cific .beta.-tuulin probe.
[0074] Drosophila Genetics: For epistasis between kuz and Notch, an
activated N construct containing only the cytoplasmic domain of
NOTCH (N )under the control of the heatshock promoter (ITM3A
insertion on the X chromosome, from Lieber, T., et al. (1993).
Gene., Dev. 7, 1949-1965) and a null kuz allele e29-4 (Rooke et
al., 1996) were used. Flies of the genotype ITM3A/+; e29-4 ck
FRT40A/+were crossed to hsFlp/Y; FRT40A. The progeny from such a
cross were subjected to a one hr heatshock at 38.degree. C 24 to 48
hrs after egg laying to induce kuz mutant clones and another one hr
heatshock at 7-9 hrs APF to induce the expression of N.sup.act.
Adult flies were processed for scanning electron microscopy and the
20 clones identified by the cell autonomous ck epidermal hair
marker as in Rooke et al. (1996). kuz germline clones were
generated as in Rooke et al. (1996). Females bearing germline
clones were mated to e29-4lCyO males. kuz null embryos lacking both
maternal and zygotic contribution can be distinguished from kuz
maternal null embryos rescued with one zygotic copy of kuz at late
embryonic stages since kuz null embryos fail to develop any cuticle
while paternally rescued embryos develop some cuticle structures.
kuz null embryos were hand-picked for making protein extracts.
[0075] Protein Extracts and Immunoblotting: About 2.times.10.sup.2
cells, 50 embryos, or imaginal discs from 16 third instar larvae
were used for each extraction. These materials were homogenized and
incubated for 20 min on ice in 90 Ill of buffer containing 10 mM
KCl, 20 30 mM Tris pH 7.5, 0.1% mercaptoethanol, I mM EDTA plus
protease and phosphatase inhibitors (leupeptin, aprotinin, PMSF and
sodium vanadate). Supernatant was collected after a low speed spin
of 2000 rpm for 5 min. 12 ,li of supernatant was run on a 6% SDS
polyacrylamide gel. Blotting, antibody incubation, and
chemiluminescent detection using the ECL kit were as described in
Fehon et al. (1990).
Sequence CWU 1
1
* * * * *