ACKNOWLEDGEMENT OF GOVERNMENT RIGHTS

This invention was made in part with Government support under Research training grant in neuropathobiology grant number 5 T32 NS07064-13 award by the National Institutes of Health. The Government has certain rights in this invention.

FIELD OF THE INVENTION The present invention relates to collagen compositions that alter tissue size, shape, and/or density and to methods of making and using the same. The present invention also relates to methods of altering the size, shape, and/or density of tissue.

BACKGROUND OF THE INVENTION

Collagen is a polypeptide substance comprising one third of the total protein in mammalian organisms. It is the main constituent of skin, connective tissue, and the organic substance of bones and teeth. Collagen production in the body is preceded by the formation of a much larger molecule, the biosynthetic precursor procollagen, which is degraded by specific enzymes to form collagen.

Different types of collagen exist. They are all composed of molecules whose predominant feature is a triple helical rod or fibrillar conformation to fill in extracellular space. The amino acid sequence of the α-chain is mostly a repeating structure with glycine in every third position and proline or 4-hydroxyproline frequently proceeding the glycine residues. Slight differences in the primary structure establish the differences between the types of collagen. Collagen is differentiated from the accompanying extracellular matrix proteins, such as, for example, elastin and reticulm,

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- 2 - by 1) its content of proline, hydroxyproline, an hydroxylysine, by 2) the absence of tryptophan and its lo tyrosine and sulfur content, but particularly by 3) its hig content of polar groups originating from the difunctional amin acids. The polar groups are responsible for the swellin properties leading eventually to dispersion of collagen i dilute acid. There is a need for compositions which alte tissue size, shape, and/or density. There is also a need fo a method of altering tissue size, shape, and/or density.

SUMMARY OF THE INVENTION

The present invention relates to a substantiall purified collagen protein that has an amino acid sequence that consists essentially of the amino acid sequence of SEQ ID NO:2. The present invention relates to an isolated nuclei acid molecule that comprises a nucleotide sequence that encode the collagen protein that has an amino acid sequence that consists essentially of the amino acid sequence of SEQ ID NO:2.

The present invention relates to an isolated nucleic acid molecule that comprises the nucleotide sequence of SEQ ID NO: 1.

The present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence identical or complementary to a fragment of SEQ ID NO: 1 which is at least 10 nucleotides in length. The present invention relates to a recombinant expression vector that comprises a nucleotide sequence that encodes the collagen protein that has an amino acid sequence that consists essentially of the amino acid sequence of SEQ ID NO:2. The present invention relates to a host cell that comprises the recombinant expression vector that comprises a nucleotide sequence that encodes the collagen protein that has an amino acid sequence that consists essentially of the amino acid sequence of SEQ ID NO: 2. The present invention relates to a transgenic mammal that comprises the recombinant expression vector that comprises

a nucleic acid sequence that encodes the collagen protein that has an amino acid sequence that consists essentially of the amino acid sequence of SEQ ID NO: 2.

The present invention relates to an injectable pharmaceutical composition that comprises a pharmaceutically acceptable carrier and the collagen protein that has an amino acid sequence that consists essentially of the amino acid sequence of SEQ ID NO:2.

The present invention relates to a method of altering tissue size, shape, and/or density in an individual by injecting into the tissue of an individual an injectable pharmaceutical composition that comprises a pharmaceutically acceptable carrier and the collagen protein that has an amino acid sequence that consists essentially of the amino acid sequence of SEQ ID NO:2.

The present invention relates to a composition that comprises the collagen protein that has an amino acid sequence that consists essentially of the amino acid sequence of SEQ ID NO: 2 free from other collagen proteins. The present invention relates to an antibody that binds to an epitope which is present on a collagen protein that has an amino acid sequence that consists essentially of the amino acid sequence of SEQ ID NO:2.

DETAILED DESCRIPTION OF THE INVENTION The present invention arises from the discovery of a new and useful member of the collagen protein family which is referred to herein as "saccular collagen". The collagen protein of the present invention is a small protein relative to other members of the collagen family. The collagen protein of the present invention contains 423 amino acids including a 217 amino acid collagenous domain having a structure characteristic of collagen family members that is in between a 57 amino acid N-terminal non-collagenous domain and a 149 amino acid C-terminal non-collagenous domain. The amino- terminal non-collagenous domain is unique to the collagen protein of the present invention while the carboxy-terminal

non-collagenous domain contains a region that is highl homologous to the carboxy terminal non-collagenous domains o the type VIII and the type X collagen.

Of all of the extracellular matrix-situated collagen with described structural roles, the collagen of the presen invention contains the smallest collagenous/triple helica domain. The supramolecular organization of saccular collage is predicated to be an extracellular, hexagonally-specified three dimensional matrix. It appears that this matrix, in th presence of the other glycoproteins associated with th otolithic membrane, resembles a fine gel.

The short chain collagens that includes collagen typ VIII and X as well as saccular collagen have the followin biomechanical properties. The three dimensional organizatio of matrices formed by the short chain collagens provide unifor tensilar strength and resistance to compressive forces compare to those formed by fibrillar collagens. Similarly, the thre dimensional organization of matrices formed by the short chai collagens provide uniform tensilar strength and resistance t compressive forces compared to either the loose random beade meshwork matrices formed by collagen VI, or the rando irregular meshwork matrices formed by collagen IV. Th matrices formed by the short chain collagens can serve as solute permeable, cell-impermeable barrier as exemplified b the formation of Descemet's membrane by collagen type VIII i the corneal endothelium. The consistency and form of matrice formed by short chain collagens can assume may be more stabl an enduring a matrix because of its unique three-dimensiona nature. The matrices formed by short chained collagen comprise a single collagenouε molecule and can for spontaneously through self assembly. Therefore, they shoul be more easily formed, maintained, and altered than comparabl compositions formed of other known collagens. Such matrice may also retain more reliable adherence to its original form The following properties distinguish matrices forme by the collagen of the present invention from those formed b oother short chain collagens, specifically the relate

collagens type VIII and X. The small size of collagenous/TK domain of saccular collagen provides a more dense lattice due to more SC domains of any kind per unit volume of matrix. The reuslt is a distinct biomechanical specification with regards to the tensilar strength and resistance to compressive forces of the matrix, i.e. there are more TH regions/unit matrix volume of saccular collagen as compared to collagen VIII or collagen X matrices. Further, there is a shorter amount of TH regions/net unit TH region in matrices of saccular collagen as compared to collagen VIII or collagen X matrices. In addition, the finer grain quality of saccular collagen make it more easily introduced in specific small regions and more easy to prepare and manipulate in large scale processing/handling.

As used herein, the term "saccular collagen" is meant to refer to the protein that has an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 2. As used herein, the term "protein that has an amino acid sequence consisting essentially of the amino acid sequence set forth in SEQ ID NO:2" is meant to refer to the saccular collagen that has an amino acid sequence that consists of the amino acid sequence set forth in SEQ ID NO:2, as well has derivative and analog proteins that have an amino acid sequence essentially identical to the amino acid sequence set forth in SEQ ID NO:2 but with conservative amino acid substitutions and/or deletions and/or insertions. Conservative amino acid sequences are well known and deletions and insertions of amino acids in a protein can be single amino acid, double amino acid or triple amino acid deletions and insertions or combinations thereof . The present invention provides a substantially purified saccular collagen protein that has an amino acid sequence consisting essentially of the amino acid sequence of SEQ ID NO:2. In particular, the present invention relates to a substantially purified saccular collagen protein that has an amino acid sequence that consists of the amino acid sequence of SEQ ID NO:2. The saccular collagen of the present invention has been isolated and purified from natural sources,

specifically from bluegill sunfish { Lepomis macrochirus) wher it is found in supporting cells located at the outer perimete of the saccular sensory epithelium.

The saccular collagen protein may be used in a variet of applications. Injectable pharmaceutical compositions ar provided which are useful in cosmetic or reconstructiv surgical procedures such as for the reduction of skin wrinkles creases, folds and the like, as well as for the enlargement o tissue such as cosmetic and reconstructive tissue implants particularly facial implants such as lip, cheek, chin and nos implants or body implants. The saccular collagen protein ma be used in cosmetic compositions and is provided i compositions free of other collagen proteins. Moreover saccular collagen protein may be used as fibers in sutures, i leather substitutes, as a gel in photographic emulsions, i coatings, and in food casings.

Saccular collagen of the invention can be isolate from natural sources, produced by recombinant DNA methods o synthesized by standard protein synthesis techniques. Antibodies which specifically bind to the saccula collagen may be used to purify the protein from natural source using well known techniques and readily available startin materials. Such antibodies may also be used to purify saccula collagen from material present when producing saccular collage by recombinant DNA methodology. The present invention relate to an antibody that binds to an epitope which is present on saccular collagen protein that comprises the amino aci sequence of SEQ ID NO:2. As used herein, the term "antibody is meant to refer to complete, intact antibodies or Fa fragments and F(ab) ₂ fragments thereof. Complete, intac antibodies include monoclonal antibodies such as murin monoclonal antibodies, chimeric antibodies and humanize antibodies. In some embodiments, the antibodies specificall bind to an epitope between amino acids 1 to 57 of SEQ ID NO: which is a unique portion of saccular collagen that constitute the N-terminal globular portion of the saccular collage molecule. In some embodiments, the antibodies specificall

bind to epitopes between amino acids 275 to 423 of SEQ ID NO: 2 which the C-terminal globular portion of the saccular collagen molecule. Preferably, the antibodies bind to epitopes in this region which are not found in corresponding regions of collagen VIII and X molecules. In some embodiments, antibodies bind to epitopes which include amino acids from the saccular collagen molecule at amino acid sequence 349-363 and/or 375- 387. Antibodies that bind to an epitope which is present on a saccular collagen protein that has SEQ ID NO: 2 are useful to isolate and purify saccular collagen that has SEQ ID NO:2 from both natural sources or recombinant expression systems using well known techniques such as affinity chromatography. Such antibodies are useful to detect the presence of such protein in a sample and to determine if cells are expressing the saccular collagen.

The production of antibodies and the protein structures of complete, intact antibodies, Fab fragments and F(ab) ₂ fragments and the organization of the genetic sequences that encode such molecules are well known and are described, for example, in Harlow, E. and D. Lane (1988) ANTIBODIES : A Labora tory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. which is incorporated herein by reference. Briefly for example, full length saccular collagen protein, or an immunogenic fragment thereof is injected into mice. The spleen of the mouse is removed, the spleen cells are isolated and fused with immortalized mouse cells. The hybrid cells, or hybridomas, are cultured and those cells which secrete antibodies are selected. The antibodies are analyzed and, if found to specifically bind to the saccular collagen, preferably the unique N-terminal portion of saccular collagen, the hybridoma which produces them is cultured to produce a continuous supply of antibodies.

Using standard techniques and readily available starting materials, a nucleic acid molecule that encodes saccular collagen may be isolated from a cDNA library, such as, for example, from bluegill sunfish saccular macula cDNA library, using probes which are designed using the nucleotide

sequence information disclosed in SEQ ID N0:1. The present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes the saccular collagen protein that comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid molecules consist of a nucleotide sequence that encodes the saccular collagen protein. In some embodiments, the nucleic acid molecules comprise the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the nucleic acid molecules consist of the nucleotide sequence set forth in SEQ ID NO: 1. The isolated nucleic acid molecules of the invention are useful to prepare constructs and recombinant expression systems for preparing the saccular collagen protein of the invention.

A cDNA library may be generated by well known techniques using supporting cells located at the outer perimeter of the saccular sensory epithelium or whole saccula macula which produce saccular collagen as starting material. cDNA made from bluegill sunfish saccular macula has been used to identify cDNA that encodes saccular collagen. It is preferred that the cDNA library be generated from supporting cells located at the outer perimeter of the saccular sensory epithelium of bluegill sunfish. For example, a cDNA clone which contains the nucleotide sequence is identified using probes that comprise at least a portion of the nucleotide sequence disclosed in SEQ ID NO: 1 having at least 16 nucleotides, preferably 24 nucleotides. It is preferred that the probes comprise all or most of the nucleotide sequence disclosed in SEQ ID NO: 1 between nucleotide 331 and nucleotide 501 of SEQ ID NO:l or nucleotide 1600 and nucleotide 1839 of SEQ ID NO: 1 and preferably no other nucleotide sequences. The probes are used to screen the cDNA library using standard hybridization techniques. Alternatively, genomic clones may be isolated using genomic DNA from any fish cell as a starting material. The present invention relates to isolated nucleic acid molecules that comprise a nucleotide seσuence identical or complementary to a fragment of SEQ ID NO: 1 which is at least 10 nucleotides. In some embodiments, the isolated nucleic acid

molecules consist of a nucleotide sequence identical or complementary to a fragment of SEQ ID NO: 1 which is at least 10 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consist of a nucleotide sequence identical or complementary to a fragment of SEQ ID NO: 1 which is 15-150 nucleotides. In some- embodiments, the isolated nucleic acid molecules comprise or consist of a nucleotide sequence identical or complementary to a fragment of SEQ ID NO:l which is 15-30 nucleotides. Isolated nucleic acid molecules that comprise or consist of a nucleotide sequence identical or complementary to a fragment of SEQ ID NO:l which is at least 10 nucleotides are useful as probes for identifying genes and cDNA sequence having SEQ ID NO: 1, PCR primers for amplifying genes and cDNA having SEQ ID NO: 1 and antisense molecules for inhibiting transcription and translation of genes and cDNA, respectively, which encode saccular collagen having the amino acid sequence of SEQ ID NO:2.

The cDNA that encodes the saccular collagen may be used as a molecular marker in electrophoresis assays in which cDNA from a sample is separated on an electrophoresis gel and saccular collagen probes are used to identify bands which hybridize to such probes. Specifically, SEQ ID NO:l, or portions thereof, may be used as a molecular marker in electrophoresis assays in which cDNA from a sample is separated on an electrophoresis gel and saccular collagen specific probes are used to identify bands which hybridize to them, indicating that the band has a nucleotide sequence complementary to the sequence of the probes. The isolated nucleic acid molecule provided as a size marker will show up as a positive band which is known to hybridize to the probes and thus can be used as a reference point to the size of cDNA that encodes saccular collagen. Electrophoresis gels useful in such an assay include standard polyacrylamide gels as described in Sambrook et al . , Molecular Cloning a Labora tory Manual , Second Ed. Cold Spring Harbor Press (1989) which is incorporated herein by reference.

The nucleotide sequence in SEQ ID NO: 1 may be used t design probes, primers and complimentary molecules whic specifically hybridize to the unique nucleotide sequences o saccular collagen. Probes, primers and complimentary molecule which specifically hybridize to nucleotide sequence tha encodes saccular collagen may be designed routinely by thos having ordinary skill in the art.

Tne present invention also includes labelle oligonucleotides which are useful as probes for performin oligonucleotide hybridization methods to identify saccula collagen. Accordingly, the present invention includes probe that can be labelled and hybridized to unique nucleotid sequences of saccular collagen. The labelled probes of th present invention are labelled with radiolabelled nucleotide or are otherwise detectable by readily available nonradioactiv detection systems. In some preferred embodiments, probe comprise oligonucleotides consisting of between 10 and 10 nucleotides. In some preferred, probes compris oligonucleotides consisting of between 10 and 50 nucleotides In some preferred, probes comprise oligonucleotides consistin of between 12 and 20 nucleotides. The probes preferabl contain nucleotide sequence completely identical o complementary to a fragment of a unique nucleotide sequence of saccular collagen. PCR technology is practiced routinely by those havin ordinary skill m the art and its uses in diagnostics are wel known and accepted. Methods for practicing PCR technology ar disclosed in "PCR Protocols: A Guide to Methods an Applications", Innis, M.A. , et al . Eds. Academic Press, Inc. San Diego, CA (1990) which is incorporated herein by reference. Applications of PCR technology are disclosed in "Poly eras Chain Reaction" Erlich, H.A., et al . , Eds. Cold Spring Haroo Press, Cold Spring Harbor, NY (1989) which is incorporate herein by reference. Some simple rules aid in the design o efficient primers. Typical primers are 18-28 nucleotides i length having 50% to 60% g+c composition. The entire prime is preferably complementary to the sequence it must hyoridiz

to. Preferably, primers generate PCR products 100 basepairs to 2000 base pairs. However, it is possible to generate products of 50 base pairs to up to 10 kb and more.

PCR technology allows for the rapid generation of multiple copies of nucleotide sequences by providing 5' and 3' primers that hybridize to sequences present in a nucleic acid molecule, and further providing free nucleotides and an enzyme which fills in the complementary bases to the nucleotide sequence between the primers with the free nucleotides to produce a complementary strand of DNA. The enzyme will fill in the complementary sequences adjacent to the primers. If both the 5' primer and 3' primer hybridize to nucleotide sequences on the complementary strands of the same fragment of nucleic acid, exponential amplification of a specific double- stranded product results. If only a single primer hybridizes to the nucleic acid molecule, linear amplification produces single-stranded products of variable length.

One having ordinary skill in the art can isolate the nucleic acid molecule that encodes saccular collagen and insert it into an expression vector using standard techniques and readily available starting materials.

The present invention relates to a recombinant expression vector that comprises a nucleotide sequence that encodes the saccular collagen protein that comprises the amino acid sequence of SEQ ID NO: 2. As used herein, the term "recombinant expression vector" is meant to refer to a plasmid, phage, viral particle or other vector which, when introduced into an appropriate host, contains the necessary genetic elements to direct expression of the coding sequence that encodes the saccular collagen of the invention. The coding sequence is operably linked to the necessary regulatory sequences. Expression vectors are well known and readily available. Examples of expression vectors include plasmids, phages, viral vectors and other nucleic acid molecules or nucleic acid molecule containing vehicles useful to transform host cells and facilitate expression of coding sequences. In some embodiments, the recombinant expression vector comprises

the nucleotide sequence set forth in SEQ ID NO: 1. Th recombinant expression vectors of the invention are useful fc transforming hosts to prepare recombinant expression system for preparing the saccular collagen protein of the invention The present invention relates to a host cell tha comprises the recombinant expression vector that comprises nucleotide sequence that encodes the collagen protein tha comprises the amino acid sequence of SEQ ID NO: 2. In som embodiments, the host cell comprises a recombinant expressio vector that comprises SEQ ID NO:1. Host cells for use in wel known recombinant expression systems for production of protein are well known and readily available. Examples of host cell include bacteria cells such as E . coli , yeast cells such as S cerevisiae, insect cells such as S . frugiptera , non-huma mammalian tissue culture cells Chinese hamster ovary (CHO cells and human tissue culture cells such as HeLa cells.

The present invention relates to a transgenic non human mammal that comprises the recombinant expression vecto that comprises a nucleic acid sequence that encodes th collagen protein that comprises the amino acid sequence of SE ID NO: 2. Transgenic non-human mammals useful to produc recombinant proteins are well known as are the expressio vectors necessary and the techniques for generating transgeni animals. Generally, the transgenic animal comprises recombinant expression vector in which the nucleotide sequenc that encodes the saccular collagen of the invention is operabl linked to a mammary cell specific promoter whereby the codin sequence is only expressed in mammary cells and the recombinan protein so expressed is recovered from the animal's milk. I some embodiments, the coding sequence that encodes the saccula collagen protein of the invention is SEQ ID NO: 1.

In some embodiments, for example, one having ordinar skill in the art can, using well known techniques, insert suc DNA molecules into a commercially available expression vecto for use in well known expression systems. For example, th commercially available plasmid pSE420 (Invitrogen, San Diego, CA) may be used for production of collagen in E. coli . Th

commercially available plasmid pYES2 (Invitrogen, San Diego, CA) may, for example, be used for production in S . cerevi siae strains of yeast. The commercially available MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, CA) may, for example, be used for production in insect cells. The commercially available plasmid pcDNA I (Invitrogen, San Diego, CA) may, for example, be used for production in mammalian cells such as Chinese Hamster Ovary cells. One having ordinary skill in the art can use these commercial expression vectors and systems or others to produce saccular collagen or fragments thereof using routine techniques and readily available starting materials. (See e . g . , Sambrook et al . , Molecular Cloning a Labora tory Manual , Second Ed. Cold Spring Harbor Press (1989) which is incorporated herein by reference.) Thus, the desired proteins can be prepared in both prokaryotic and eukaryotic systems, resulting in a spectrum of processed forms of the protein.

One having ordinary skill in the art may use other commercially available expression vectors and systems or produce vectors using well known methods and readily available starting materials. Expression systems containing the requisite control sequences, such as promoters and polyadenylation signals, and preferably enhancers, are readily available and known in the art for a variety of hosts. See e . g . , Sambrook et al . , Molecular Cloning a Labora tory Manual , Second Ed. Cold Spring Harbor Press (1989) .

The most commonly used prokaryotic system remains E . coli , although other systems such as B . subtilis and Pseudomonas are also useful. Suitable control sequences for prokaryotic systems include both constitutive and inducible promoters including the lac promoter, the crp promoter, hybrid promoters such as tac promoter, the lambda phage Pi promoter. In general, foreign proteins may be produced in these hosts either as fusion or mature proteins. When the desired sequences are produced as mature proteins, the sequence produced may be preceded by a methionine which is not necessarily efficiently removed. Accordingly, the peptides ana

proteins claimed herein may be preceded by an N-terminal Me when produced in bacteria. Moreover, constructs may be mad wherein the coding sequence for the peptide is preceded by a operable signal peptide which results in the secretion of th protein. When produced in prokaryotic hosts in this matter the signal sequence is removed upon secretion.

A wide variety of eukaryotic hosts are also no available for production of recombinant foreign proteins. A in bacteria, eukaryotic hosts may be transformed wit expression systems which produce the desired protein directly but more commonly signal sequences are provided to effect th secretion of the protein. Eukaryotic systems have th additional advantage that they are able to process intron which may occur in the genomic sequences encoding proteins o higher organisms. Eukaryotic systems also provide a variet of processing mechanisms which result in, for example glycosylation, carboxy-terminal amidation, oxidation o derivatization of certain amino acid residues, conformationa control, and so forth. Commonly used eukaryotic systems include, but is no limited to, yeast, fungal cells, insect cells, mammalian cells avian cells, and cells of higher plants. Suitable promoter are available which are compatible and operable for use in eac of these host types as well as are termination sequences an enhancers, e . g . the baculovirus polyhedron promoter . As above promoters can be either constitutive or inducible. Fo example, in mammalian systems, the mouse metallothionei promoter can be induced by the addition of heavy metal ions.

The particulars for the construction of expressio systems suitable for desired hosts are known to those in th art. Briefly, for recombinant production of the protein, th DNA encoding the polypeptide is suitably ligated into th expression vector of choice. The DNA is operably linked to al regulatory elements which are necessary for expression of th DNA in the selected host. One having ordinary skill in the ar can, using well known techniques, prepare expression vector for recombinant production of the polypeptide.

The expression vector including the DNA that encodes saccular collagen or a fragment thereof is used to transform the compatible host which is then cultured and maintained under conditions wherein expression of the foreign DNA takes place. The protein of the present invention thus produced is recovered from the culture, either by lysing the cells or from the culture medium as appropriate and known to those in the art . One having ordinary skill in the art can, using well known techniques, isolate saccular collagen or fragment that is produced using such expression systems. The methods of purifying saccular collagen from natural sources using antibodies which specifically bind to saccular collagen as described above, may be equally applied to purifying saccular collagen produced by recombinant DNA methodology. Examples of genetic constructs useful for transfecting with cells with DNA that encodes full length saccular collagen in order to express full length saccular collagen include SEQ ID NO:l operably linked to a promoter that is functional in the cell line into which the constructs are transfected. Examples of constitutive promoters include promoters from cytomegalovirus or SV40. Examples of inducible promoters include mouse mammary leukemia virus or metallothionein promoters. Those having ordinary skill in the art can readily produce genetic constructs useful for transfecting with cells with DNA that encodes full length saccular collagen from readily available starting materials. Such gene constructs are useful for the production of saccular collagen protein.

In some embodiments of the invention, transgenic non- human animals are generated. The transgenic animals according to the invention contain SEQ ID NO: 1 under the regulatory control of a mammary specific promoter. One having ordinary skill in the art using standard techniques, such as those taught in U.S. Patent No. 4,873,191 issued October 10, 1989 to Wagner and U.S. Patent No. 4,736,866 issued April 12, 1988 to Leder, both of which are incorporated herein by reference, can produce transgenic animals which produce the full length

saccular collagen. Preferred animals are rodents, particularl goats, rats and mice.

In addition to producing these proteins by recombinan techniques, automated peptide synthesizers may also be employe to produce saccular collagen or fragments. Such techniques ar well known to those having ordinary skill in the art and ar useful if derivatives which have substitutions not provided fo in DNA-encoded protein production.

Pharmaceutical compositions according to the inventio comprise a pharmaceutically acceptable carrier in combinatio with saccular collagen protein or fragment. Pharmaceutica formulations for injectable collagen are well known an pharmaceutical compositions comprising saccular collagen ma be routinely formulated by one having ordinary skill in th art. Suitable pharmaceutical carriers are described i Remington ' s Pharmaceutical Sciences , A. Osol, a standar reference text in this field, which is incorporated herein b reference. The present invention relates to an injectabl pharmaceutical composition that comprises a pharmaceuticall acceptable carrier and the collagen protein that has the amin acid sequence of SEQ ID NO:2. Some embodiments of th invention relate to injectable pharmaceutical compositions tha comprise a pharmaceutically acceptable carrier and the saccula collagen protein that has an amino acid sequence that consist of SEQ ID NO:2. As discussed above, the saccular collagen of the invention may be injected into the tissue of an individual for cosmetic and/or reconstructive surgical purposes. Suc injections effectively alter the size, shape and/or density o the tissue. The saccular collagen is sterile and combined wit a sterile pharmaceutical carrier.

In some embodiments, for example, saccular collage can be formulated as a solution, suspension, emulsion o lyophilized powder in association with a pharmaceuticall acceptable subcutaneous vehicle. Examples of such vehicles ar water, saline, Ringer's solution, dextrose solution, and 5 human serum albumin. Liposomes and nonaqueous vehicles suc as fixed oils may also be used. The vehicle or lyophilize

powder may contain additives that maintain isotonicity (e.g. , sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives) . The formulation is sterilized by commonly used techniques. For example, a subcutaneous composition suitable for administration by injection is prepared by dissolving 1.5% by weight of saccular collagen in 0.9% sodium chloride solution.

An injectable composition may comprise saccular collagen in a diluting agent such as, for example, sterile water, electrolytes/dextrose, fatty oils of vegetable origin, fatty esters, or polyols, such as propylene glycol and polyethyleneglycol . The injectable must be sterile and free of pyrogens .

The pharmaceutical compositions of the present invention may be administered to an individual suffering from a variety of skin or connective tissue disorders so that tissue size, shape, and/or density will be affected. The present invention relates to a method of altering tissue size, shape, and/or density in an individual by injecting into the tissue of an individual an injectable pharmaceutical composition that comprises a pharmaceutically acceptable carrier and the saccular collagen protein that has an amino acid that consists of SEQ ID NO:2. Examples of tissue where saccular collagen may be injected include skin which is wrinkled, creased or folded, such as skin around the eyes and mouth. Saccular collagen may be injected into an individual's lips, nose, chin or cheeks. Surgical procedures for collagen injection and implantation are well known.

The present invention relates to a composition that comprises the saccular collagen protein that have an amino acid seσuence that consists of SEQ ID NO:2 free from other collagen proteins. Examples of such compositions include drug delivery systems, cosmetics and foods. The saccular collagen may be combined with drugs in a drug delivery system or device including ointments, lotions and the like as well as sustained release/time release compositions. In some embodiments, the saccular collagen is combined with other compounds to produce

cosmetic creams, oils, gels, powders and the like. In som embodiments, the saccular collagen is combined with food stuff as a food ingredient.

Topical compositions preferably include lotions whic may contain numerous compounds in addition to saccula collagen. Bentonite, as well as other suitable substitutes may be included as a suspension agent. Methylcellulose o sodium carboxymethylcellulose may be included to localize an hold saccular collagen in contact with the affected site Glycerin may be included to keep the skin moist. Alcohol ma be included to aid in drying and cooling. Preservatives ma be included to inhibit microbial growth. Fragrances may als be included.

EXAMPLES Example 1: Construction of cDNA Libraries

The saccular sensory epithelium in an adult bluegil sunfish { Lepomis macrochirus) may contain as many as, or more than 2 x 10 ^s hair and supporting cells (Corwin, J. Comp . Neur. 1981, 201:541) . Oligo-dT and random primed cDNA libraries wer constructed from poly-A+ selected mRNAs . The oligo-dt prime library was constructed in λgtll and consisted of nearly 9. x 10 ^s recombinants ranging between 0.3 and 3.5 kB in length. Example 2: Differential Screening of cDNA Libraries

Bluegill sunfish saccular macula cDNA libraries wer differentially screened to identify inner ear-specifi transcripts. Differential screening of an oligo-dT prime saccular macula cDNA library with labeled saccular macula an liver A+ cDNAs was performed. Briefly, differential screenin was performed using ³² P labeled sunfish saccular maculae an liver cDNAs that were prepared from poly-A+ RNAs and/o amplified RNAs (VanGelder et al . , Proc. Acad . Sci . U. S . A . 1990, 87:1663) using standard protocols (Maniatis et al . Mol ecular Cloning : A Labora tory Manual , Cold Spring Harbo Laboratory, Cold Spring Harbor, NY (1982)) . First strand cDN was synthesized from a given A+ RNA or aRNA aliquot, wa purified, and was then used as template in a standard rando

primed reaction to generate labeled cDNA probe. Recovery was on the order of lO ^{^} cpm/100 ng of any of the starting RNAs per reaction. Nitrocellulose lifts (Schleicher and Schuell, Keene, NH) of the saccular maculae cDNA library platings were hybridized with labeled probe (10 cpm/ml) in 6X SSC/0.1% SDS/0.05X. These lifts were hybridized overnight at 60°C. After high stringency washes, the hybridized lifts were subjected to autoradiography at -70°C. Plaques corresponding to hybridization signals derived from the macula-probed lifts for a given plate that did not match with a corresponding signal on the liver-probed lifts from the corresponding plate were isolated from the library platings. cDNA inserts were isolated from purified phage preparations and ligated into pBluescript (Stratagene Cloning Systems, La Jolla, CA) according to standard cloning protocols (Maniatis et al . , SUPRA) . Sanger dideoxy chain termination sequencing was performed using the Sequencing Version 2.0 DNA Sequencing system (United States Biochemical Corp., Cleveland, Ohio) . The cDNA was sequenced nearly in its entirety on both strands. Nucleotide and amino acid sequence analysis was performed with MacVector 4.0 software. Homology searches were performed with Blast search programs (Altschul et al . , J. Mol . Biol . , 1990, 215:403) .

The differential expression of the first saccule- specific cDNA was examined by northern analysis. Northern blot analysis was performed with a labeled 240 nucleotide 3' end- derived probe. Total RNAs were prepared using guanidium isothiocyanate tissue lysis followed by acid phenol extraction (Chomczynaki and Sacchi, Anal. Biochem . , 1987, 162 : 156 ) . Northern analysis was performed using 6% formaldehyde-1. 96 agarose denaturing gel electrophoresis was performed as described in Maniatis et al . ( SUPRA) . RNAs were capillarily transferred and cross-linked to Zeta-Probe nylon membranes and were then prehybridized, hybridized, and washed according to the membrane manufacturer's specifications (Biorad Chemical Division, Richmond, CA) ) and yielded a panel of macula-specific clones. The message corresponding to this cDΝA was identified

in 1 μg of sunfish saccular macula (SM) total RNA as a singl transcript nearly 2.0 kb in length. A similar message was no detected in 2 μg samples of sunfish gill (G) , heart (H) , par superior (PS = vestibular portion of ear) , retina (R) , brai (B) , or liver (L) . The blot was stripped and rehybridized t a cytoplasmic S- ctin probe to confirm RNA integrity. I addition, no detectable expression of this gene was observe in 1 μg of poly-A+ RNA from sunfish brain, liver, or muscle. Example 3 : Analysis of the Saccular collagen Gene The complete nucleotide as well as the predicted amin acid sequence corresponding to the primary open reading fram of the cDNA have been determined (SEQ ID NO:l) . The translate amino acid sequence of the 1269 nucleotide primary open readin frame (shown beneath the nucleic acid sequence and in SEQ I NO:2) was found to encode a 423 amino acid structural protei containing a single 217 amino acid collagenous domain (amin acid positions 58-274 flanked by a 57 amino acid amino-termina non-collagenous domain and a 149 amino acid carboxy-termina non-collagenous domain. The collagenous domain contains 7 Gly-X-Y repeats with two minor imperfections (one Gly-X-Y-X-Y Gly-X-Y and one Gly-X-Gly-X-Y) and has a 12 proline and an 8 lysine content. One potential N-linked glycosylation site i identified in each non-collagenous domain. The carboxy terminal non-collagenous domain contains a region that i highly homologous to the carboxy terminal non-collagenou domains of the type VIII and the type X collagen. The 95 amin acid representing the C-terminal non-collagenous domain wa found to contain an average of 40% identity and 56% overal homology at the amino acid level with the type VIII and typ X sequences in any of the species in which these genes hav been identified. This region of homology is also shared wit the Clq complement proteins (Reid and Day, Immunol . Today 1990, 11:387; Brass et al . , Febs Lett . , 1992, 303:126) . Th amino-terminal non-collagenous domain displays no homology wit any sequences reported to date. The first 19 amino acids o the amino terminal non-collagenous domain represent a putativ signal peptide.

Collagen type X and VIII (Yamaguchi et al . , J. Biol . Che . , 1991, 266:4508; Yamagiuchi et aJ . , J. Biol . Chem . , 1989, 264 : 16022; Ninomlya et al . , J. Biol . Chem . , 1986, 262:5041; Muragaki et al . , J. Biol . Chem . , 1991, 266:7721; and LuValle et al., J. Biol . Chem . , 1988, 263:18378) and this saccular collagen are distinguished by several features. First, they share a common domain organization: a single, relatively short collagenous domain separating small amino and carboxy terminal non-collagenous (NC) globular domains resulting in a dumbbell shaped molecule. They also share the related stretch of amino acids in each of their respective carboxy terminal NC domains mentioned above. In addition, each possesses a unique amino terminal NC domain that is not related to any sequences reported to date. This macular collagen is, therefore, a novel member of this family of short chain vertebrate collagen. The most distinguishing feature of this macular collagen is that it possesses a collagenous domain of only about half of the length of the collagenous domains in the type VIII and X collagen. Example 4: In Situ Hybridization

Non radioactive in si tu hybridization was used to deduce the localization of this gene's expression within the sunfish saccular epithelium. Serial 5 micron thick sections of Bouin' s-fixed, paraffin-embedded saccular maculae were pre- treated, hybridized with antisense and sense digoxigenin labeled RNA probes specific to portions of the 3' end of the gene (none of the collagen domain coding sequence was included) .

The resultant DIG-RNA/tissue mRNA hybrids were detected using an alkaline phosphatase conjugated anti- digoxigenin antibody. A cDΝA containing 585 nucleotides of the 31 end of the macular collagen was obtained and band purified. The more 5' of the two fragments obtained by Sad digestion of this fragment was cloned into EcoRI/SacI digested pBluescript (Stratagene Cloning Systems, La Jolla, CA) . Microgram quantities of a construct containing this fragment (none of the collagen encoding domain was included) were linearized to

completion separately with an appropriate restriction enz that digested t each end of the cDNA insert. Saci digest versions were treated with Klenow to create blunt ends at the 3' overhangs before riboprobe synthesis. All restriction a modifying enzymes were from BMB, Inc. and were used accordi to standard protocols (Maniatis et al . , SUPRA) . Digoxigen (DIG) -labeled RNAs were synthesized using the Genius Labeli kit (Boehringer Mannheim Biochemical, Inc. (BMB) , Indianapoli IN) . Non-radioactive in situ hybridization was performed usi the Genius Nonradioactive Labeling and Detection System (BM Inc., Indianapolis, IN) . Details of in si tu hybridizati protocols not itemized were closely parallel than tho previously detailed (Simmons et al . , J. Hi s o technology, 198 22:169) , whereas optimization of the immunodetection of DI RNA/tissue RNA hybrids were based on several versions alrea reported for use with nonradioactive probes (Fisher et al . , Dermatol . , 1991, 125:516; Springer et al . , J. Histoche Cytoche . , 1991, 39:231) . Retained DIG-RNA/tissue cDNA hybri were detected using alkaline phosphatase conjugated ant digoxigenin Fab fragments and X-phosphate/NBT substrate bas colori etric development according to the manufacturer specifications as previously optimized (Fisher et al . , Dermatol., 1991, 125:516) . The in si tu hybridizations may photographed at approximately 100-200 X using 100-ASA Kod color film. Transcripts encoding this protein were localiz to the edges of the saccular epithelium whereas sectio hybridized with a control probe showed only light, diffu background staining. The corresponding hematoxylin and eos stained section indicated that this expression was restrict to a group of specialized secretory supporting cells th reside around the edges of the saccular epithelium. Anoth histologic section is shown in which the columnar, margin zone supporting cells.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Greene, Mark I. Davis, James G. (ii) TITLE OF INVENTION: Saccular collagen and Compositions and Methods for Making and Using the Same (iii) NUMBER OF SEQUENCES: 2 <iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Woodcock Washburn Kurtz Mackiewicz & Norris

(B) STREET: One Liberty Place, 46th floor

(C) CITY: Philadephia

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19103

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: WordPerfect 5.1 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/383,744

(B) FILING DATE: 02-FEB-1995

(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: DeLuca, Mark

(B) REGISTRATION NUMBER: 33,229

(C) REFERENCE/DOCKET NUMBER: UPN-2653 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-568-3100

(B) TELEFAX: 215-568-3439

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1839 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 331..1602

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :

CCTCTGAAGG TTCCAGAATC GATAGTGAAT TCGTGGACTA GTTGGAGAGG ATGGAATGAA 60

GAGTCAACAC AGGCTACACC TGCAGATTAT TGTGTCAAGC CCTCAGTATG CTGTCCATTC 120

GTCTAGTCTT CCTGACTGCT CTTCTTGTGG TACTGATGGC TGTGCTGACC TCCAGCACCA 180

GAACCACACG ATGGCCCAAA CCTCAGACAA CCAAGAAGCC TCCTCGAGCT GGGAGCAGCG 240

TGGAGGTGGG GTGGAGGATT CAAACGGACC ACTACCACCA CCCCATCTCC TACCAGTAGC 300

CTGCACACAG ACGAGACAAC TGAGGTTATG ATG GAC GCT TAC TCC TTG TCC CCT 354

Met Asp Ala Tyr Ser Leu Ser Pro 1 5

ACA GAC AGC ACC ACC TAC TCC AGC GAC ACT TTC TCC ACC GAG TTC CAC 402 Thr Asp Ser Thr Thr Tyr Ser Ser Asp Thr Phe Ser Thr Glu Phe His 10 15 20

ACC GAT GCC ATA GCG CCC CCT GGC AAC ACC CCT GGA AAC TAT ACC CTT 45

Thr Asp Ala lie Ala Pro Pro Gly Asn Thr Pro Gly Asn Tyr Thr Leu 25 30 35 40

GAT TAT AAT GAA TGC TTC TTC AAC TTC TGT GAG TGC TGT CCA CCA GAG 49

Asp Tyr Asn Glu Cys Phe Phe Asn Phe Cys Glu Cys Cys Pro Pro Glu 45 50 55

AAA GGC CCC ATG GGG CCC ATG GGA GAG AGA GGG CTG CCA GGA CCG CCA 54

Lys Gly Pro Met Gly Pro Met Gly Glu Arg Gly Leu Pro Gly Pro Pro 60 65 70

GGA GAG AGG GGT CCT CTA GGG TTA CCA GGG GAG AAG GGA GAG ACA GGG 59

Gly Glu Arg Gly Pro Leu Gly Leu Pro Gly Glu Lys Gly Glu Thr Gly 75 80 85

CTC AGA GGA CCT CCA GGA CCA GCA GGT CTA CCT GGA GCC AAT GGA CTC 64

Leu Arg Gly Pro Pro Gly Pro Ala Gly Leu Pro Gly Ala Asn Gly Leu 90 95 100

AAT GGC GAC ATA GGT GAA AAA GGT GAT CAA GGA CCG GTG GGT CTT CCT 69

Asn Gly Asp lie Gly Glu Lys Gly Asp Gin Gly Pro Val Gly Leu Pro 105 110 115 120

GGT GTC CCT GGG ATC CCA GGA AAA CCA GGA GAG AAA GGT GAT CCA GGC 73

Gly Val Pro Gly lie Pro Gly Lys Pro Gly Glu Lys Gly Asp Pro Gly 125 130 135

CTC AAA GGA GAT AAA GGT GAA CGT GGC TTC AGT GGT CTG AAA GGG GAC 786

Leu Lys Gly Asp Lys Gly Glu Arg Gly Phe Ser Gly Leu Lys Gly Asp

140 145 150

CCG GGA GAA AGA GGA GAG CCT GGC CTA AAT GGA ACT AAA GGA AGC ATC 834

Pro Gly Glu Arg Gly Glu Pro Gly Leu Asn Gly Thr Lys Gly Ser lie 155 160 165

GGG CGA GAG GGG CCC ATG GGT CCT GGG TTA GCT GGG ACA AAG GGT CTG 882

Gly Arg Glu Gly Pro Met Gly Pro Gly Leu Ala Gly Thr Lys Gly Leu 170 175 180

AAA GGT GAA CAG GGG CTT AAA GGC GAG TGT TTA CAA GGC GAG AAA GGT 93

Lys Gly Glu Gin Gly Leu Lys Gly Glu Cys Leu Gin Gly Glu Lys Gly 185 190 195 200

GAG CGT GGG CCC CCT GGT TTG AGA GGT GAG ATG GGA TTG AAT GGA ACT 978

Glu Arg Gly Pro Pro Gly Leu Arg Gly Glu Met Gly Leu Asn Gly Thr 205 210 215

GAT GGT GTA AAG GGA GAG AGA GGG GAG CCA GGG CCT CTT GGA GGG AAG 1026

Asp Gly Val Lys Gly Glu Arg Gly Glu Pro Gly Pro Leu Gly Gly Lys

220 225 230

GGG GAT ACT GGT GCC AGA GGG CCC CCA GGT CCT CCA GGA GGG AGG GGC 1074

Gly Asp Thr Gly Ala Arg Gly Pro Pro Gly Pro Pro Gly Gly Arg Gly 235 240 245

ATG GCA GGG TTG AGG GGG GAG AAG GGG CTT AAA GGT GTG CGT GGG CCA 1122

Met Ala Gly Leu Arg Gly Glu Lys Gly Leu Lys Gly Val Arg Gly Pro 250 255 260

AGG GGC CCT AAA GGC CCA CCA GGT GAG AGT GTG GAG CAG ATT CGC TCT 117

Arg Gly Pro Lys Gly Pro Pro Gly Glu Ser Val Glu Gin lie Arg Ser 265 270 275 280

GCT TTC AGT GTG GGC TTG TTC CCG AGC AGA TCC TTC CCT CCG CCC AGC 1218

Ala Phe Ser Val Gly Leu Phe Pro Ser Arg Ser Phe Pro Pro Pro Ser 285 290 295

CTG CCT GTG AAG TTT GAT AAG GTG TTT TAC AAC GGG GAG GGG CAC TGG 1266 Leu Pro Val Lys Phe Asp Lys Val Phe Tyr Asn Gly Glu Gly His Trp 300 305 310

GAC CCA ACA CTC AAC AAA TTC AAT GTC ACC TAC CCG GGG GTC TAC CTA 1314 Asp Pro Thr Leu Asn Lys Phe Asn Val Thr Tyr Pro Gly Val Tyr Leu 315 320 325

TTC AGT TAC CAC ATC ACC GTG CGC AAC AGG CCT GTG CGT GCT GCC CTA 1362 Phe Ser Tyr His lie Thr Val Arg Asn Arg Pro Val Arg Ala Ala Leu 330 335 340

GTG GTT AAT GGG GTA CGG AAG CTG AGG ACC CGG GAT TCT CTG TAC GGC 1410 Val Val Asn Gly Val Arg Lys Leu Arg Thr Arg Asp Ser Leu Tyr Gly 345 350 355 360

CAG GAC ATC GAT CAG GCG TCC AAC CTC GCA CTG CTG CAT CTG ACT GAC 1458 Gin Asp lie Asp Gin Ala Ser Asn Leu Ala Leu Leu His Leu Thr Asp 365 370 375

GGT GAC CAG GTG TGG CTG GAG ACA CTG AGA GAC TGG AAT GGA GTT ACT 1506 Gly Asp Gin Val Trp Leu Glu Thr Leu Arg Asp Trp Asn Gly Val Thr 380 385 390

CCA GCA GTG AGG ATG ACA GCA CTT TCT CTG GCT TCT TGC TTT ACC CTG 1554 Pro Ala Val Arg Met Thr Ala Leu Ser Leu Ala Ser Cys Phe Thr Leu 395 400 405

ACA CAA AGA AAC CTA CTG CTA TGG AAA ACC TGT GAA GGC AAA CTT TAA 1602 Thr Gin Arg Asn Leu Leu Leu Trp Lys Thr Cys Glu Gly Lys Leu 410 415 420

CCTTGAATGA GCTCTGATTG TAACTTCCTG TATACTCTGC ACAACCTTCA GCCTATTGCA 1662

CTGCTCTGTG AAATTAATGT GTTTCAGCTT AATACTGCTG CTTCAGTATC TATCGTGTTA 1722

ATCAAACCAG AATGCTTTGC TTGCCCTACT GTATTTGAAG ATGGACAAGG CTTGATTATA 1782

GTGCTGTATG CAACCCTTCA GTTCTTTGTA ACAATGCATT AAAAATGACT ACGCTTG 1839

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

Met Asp Ala Tyr Ser Leu Ser Pro Thr Asp Ser Thr Thr Tyr Ser Ser 1 5 10 15

Asp Thr Phe Ser Thr Glu Phe His Thr Asp Ala lie Ala Pro Pro Gly 20 25 30

Asn Thr Pro Gly Asn Tyr Thr Leu Asp Tyr Asn Glu Cys Phe Phe Asn 35 40 45

Phe Cys Glu Cys Cys Pro Pro Glu Lys Gly Pro Met Gly Pro Met Gly 50 55 60

Glu Arg Gly Leu Pro Gly Pro Pro Gly Glu Arg Gly Pro Leu Gly Leu 65 70 75 80

Pro Gly Glu Lys Gly Glu Thr Gly Leu Arg Gly Pro Pro Gly Pro Ala 85 90 95

Gly Leu Pro Gly Ala Asn Gly Leu Asn Gly Asp lie Gly Glu Lys Gly 100 105 110

Asp Gin Gly Pro Val Gly Leu Pro Gly Val Pro Gly lie Pro Gly Lys 115 120 125

Pro Gly Glu Lys Gly Asp Pro Gly Leu Lys Gly Asp Lys Gly Glu Arg 130 135 140

Gly Phe Ser Gly Leu Lys Gly Asp Pro Gly Glu Arg Gly Glu Pro Gly 145 150 155 160

Leu Asn Gly Thr Lys Gly Ser lie Gly Arg Glu Gly Pro Met Gly Pro 165 170 175

Gly Leu Ala Gly Thr Lys Gly Leu Lys Gly Glu Gin Gly Leu Lys Gly 180 185 190

Glu Cys Leu Gin Gly Glu Lys Gly Glu Arg Gly Pro Pro Gly Leu Arg 195 200 205

Gly Glu Met Gly Leu Asn Gly Thr Asp Gly Val Lys Gly Glu Arg Gly 210 215 220

Glu Pro Gly Pro Leu Gly Gly Lys Gly Asp Thr Gly Ala Arg Gly Pro 225 230 235 240

Pro Gly Pro Pro Gly Gly Arg Gly Met Ala Gly Leu Arg Gly Glu Lys 245 250 255

Gly Leu Lys Gly Val Arg Gly Pro Arg Gly Pro Lys Gly Pro Pro Gly 260 265 270

Glu Ser Val Glu Gin lie Arg Ser Ala Phe Ser Val Gly Leu Phe Pro 275 280 285

Ser Arg Ser Phe Pro Pro Pro Ser Leu Pro Val Lys Phe Asp Lys Val 290 295 300

Phe Tyr Asn Gly Glu Gly His Trp Asp Pro Thr Leu Asn Lys Phe Asn 305 310 315 320

Val Thr Tyr Pro Gly Val Tyr Leu Phe Ser Tyr His lie Thr Val Arg 325 330 335

Asn Arg Pro Val Arg Ala Ala Leu Val Val Asn Gly Val Arg Lys Leu 340 345 350

Arg Thr Arg Asp Ser Leu Tyr Gly Gin Asp lie Asp Gin Ala Ser Asn 355 360 365

Leu Ala Leu Leu His Leu Thr Asp Gly Asp Gin Val Trp Leu Glu Thr 370 375 380

Leu Arg Asp Trp Asn Gly Val Thr Pro Ala Val Arg Met Thr Ala Leu 385 390 395 400

Ser Leu Ala Ser Cys Phe Thr Leu Thr Gin Arg Asn Leu Leu Leu Trp 405 410 415

Lys Thr Cys Glu Gly Lys Leu 420