Recombinant Lubricin Molecules And Uses Thereof

Abstract

Recombinant lubricin molecules and uses thereof. Novel recombinant lubricin molecules and their uses as lubricants, anti-adhesive agents and/or intra-articular supplements for, e.g., synovial joints, meniscus, tendon, peritoneum, pericardium and pleura, are provided.

Description

[0001] The invention relates to novel recombinant lubricin molecules and their uses as lubricants, anti-adhesive agents and/or intra-articular supplements for, e.g., synovial joints, meniscus, tendon, peritoneum, pericardium and pleura.

BACKGROUND OF THE INVENTION

[0002] Optimal functionality of synovial joints is dependent upon extremely low coefficients of friction between articulating tissues. Normally, a contiguous, well-lubricated surface is maintained on articular cartilage. During osteoarthritis (OA), however, reduced lubrication contributes to cartilage matrix degradation and fibrillation; these in turn contribute to joint dysfunction and pain. Reduced lubrication also leads to joint dysfunction and pain in other forms of arthritis, including rheumatoid arthritis (RA).

[0003] For other tissues (e.g., tendons), a lubricated surface also contributes to optimal functionality. In addition to requiring a lubricated surface, normal tendon function requires the prevention of cellular adhesion to tendon surfaces. In flexor tendon injury and repair, for example, the formation of tendon adhesions is the most common complication.

[0004] Native lubricin protein is related to megakaryocyte stimulating factor (MSF) precursor protein. PRG4 (proteoglycan 4) is the name for MSF that has been accepted for the UCL/HGNC/HUGO Human Gene Nomenclature database. PRG4 protein (i.e., the MSF precursor protein) is described in U.S. Pat. No. 6,433,142 and US20020137894 (all patents and patent applications cited in this document are incorporated by reference in their entirety). Polypeptide encoded by exon 6 of the PRG4 gene is heavily glycosylated and appears necessary for a PRG4-related protein to serve as a lubricant, e.g., between surfaces of articular cartilage.

[0005] Studies indicate that PRG4 glycoprotein is also synthesized by the intimal synoviocytes that line tendon sheaths; it is highly likely that the glycoprotein also originates from tenocytes (Rees et al., 2002). The glycoprotein is prominently present in fibrocartilaginous regions of tendon. In a manner complementary to its synovial-fluid function, the glycoprotein may play an important cytoprotective role for tendons by preventing cellular adhesion to tendon surfaces, as well as by providing lubrication during normal tendon function.

[0006] Exon 6 of the PRG4 (also called "lubricin") gene encodes approximately 76-78 repeats of KEPAPTT-similar sequences and 6 repeats of XXTTTX-like sequences. Varying the number of comparable repeat sequences in recombinant lubricin proteins according to the present invention allows for development of improved biotherapeutics for enhancing lubrication in joints and for countering undesired adhesion between tissues.

SUMMARY OF THE INVENTION

[0007] The present invention relates to novel recombinant lubricin molecules and their use as lubricants, anti-adhesive agents and/or intra-articular supplements.

[0008] In order to optimize expression parameters and investigate the functional necessity of all approximately 76-78 KEPAPTT-similar sequences, lubricin expression constructs were designed which enabled the synthesis of recombinant lubricin proteins with varying degrees of O-linked oligosaccharide substitution. This is accomplished by incorporating variable numbers of the KEPAPTT-like sequences into a "core" cDNA construct comprised of exons 1 through 5, 5'- and 3'-flanking regions of exon 6, and exons 7 through 12. Iterative insertion of "synthetic cDNA cassettes" encoding multiple KEPAPTT-like sequences facilitates the generation of recombinant lubricin constructs of different sizes. The initial focus of these studies was on construct PRG4-Lub:1 (containing DNA of "synthetic cDNA cassette-1" (SEQ ID NO: 1), which encodes four KEPAPTT sequences).

[0009] The recombinant lubricin proteins of the present invention share primary structure with several isoforms of native human lubricin (see U.S. Pat. No. 6,743,774, US20040072741, and WO0064930). Among characterized isoforms, each isoform differs in the composition of PRG4 gene exons that encode the isoform's primary structure. For example, exons 1 through 12 of the PRG4 gene encode the V0 isoform, which represents the full-length isoform, while exons 1 through 4 and 6 through 12 encode the V1 isoform, which lacks only a segment encoded by exon 5. Exons 1 through 3 and 6 through 12 encode the V2 isoform, which lacks segments encoded by exons 4 and 5. Finally, exons 1, 3, and 6 through 12 encode the V3 isoform, which lacks segments encoded by exons 2, 4, and 5. Other isoforms likely exist, and some related mutant proteins have been described (see US20020086824).

[0010] In particular, the present invention provides recombinant lubricin protein comprising repetitive KEPAPTT-like sequences. In preferred embodiments, the invention provides isolated protein comprising SEQ ID NOS: 9, 13, 17, 21 or 25. The invention provides in related embodiments isolated protein comprising SEQ ID NOS: 7, 11, 15, 19 or 23. In further related embodiments, the invention provides isolated polynucleotide comprising nucleic acid sequence encoding recombinant lubricin protein. In preferred embodiments, the invention provides isolated polynucleotide comprising nucleic acid sequence encoding the protein. In further related embodiments, the invention provides isolated polynucleotide having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ ID NOS: 6, 10, 14, 18 or 22 over the entire length of the sequence.

[0011] In related aspects, the present invention also provides an isolated protein comprising SEQ ID NO: 26 joined to (N minus 2) repeat(s) of SEQ ID NO: 27, where N equals an integer from 3 through 200. In further related embodiments, the present invention provides an isolated protein comprising SEQ ID NO: 26 plus SEQ ID NO: 28 plus [(N minus 2) repeat(s) of SEQ ID NO: 27] plus SEQ ID NO: 29, where N equals an integer from 3 through 200. In embodiments of the related aspects of the invention noted in this paragraph, more preferably N equals an integer from 5 through 50, and even more preferably N equals an integer from 10 through 30.

TABLE-US-00001 TABLE 1 Identification of Sequences Having Sequence Identifiers SEQ ID NO: Identification 1 nucleotide sequence of synthetic cDNA cassette-1: 155 bases 2 translation of SEQ ID NO: 1: 51 amino acids 3 nucleotide sequence of synthetic cDNA cassette-2: 125 bases 4 translation of SEQ ID NO: 3: 41 amino acids 5 pTmed2 vector containing recombinant PRG4-Lub: 1 cDNA construct: 8049 bases 6 recombinant PRG4-Lub: 1 cDNA construct: 2946 bases 7 amino acid sequence of entire PRG4-LUB: 1 protein: 981 amino acids 8 Lub: 1 DNA insert from synthetic cDNA cassette-1: 157 bases 9 51 amino acids encoded by Lub: 1 DNA insert (4 KEPAPTT sequences between S373 to E425 in SEQ ID NO: 7) 10 recombinant PRG4-Lub: 2 cDNA construct: 3024 bases 11 amino acid sequence of entire PRG4-LUB: 2 protein: 1007 amino acids 12 Lub: 2 DNA insert from synthetic cDNA cassette-1 and one synthetic cDNA cassette-2 sequence: 235 bases 13 77 amino acids encoded by Lub: 2 DNA insert (6 KEPAPTT sequences between S373 and E451 in SEQ ID NO: 11) 14 recombinant PRG4-Lub: 3 cDNA construct: 3117 bases 15 amino acid sequence of entire PRG4-LUB: 3 protein: 1038 amino acids 16 Lub: 3 DNA insert from synthetic cDNA cassette-1 and two synthetic cDNA cassette-2 sequences: 328 bases 17 108 amino acids encoded by Lub: 3 DNA insert (9 KEPAPTT sequences between S373 and E482 in SEQ ID NO: 15) 18 recombinant PRG4-Lub: 4 cDNA construct: 3210 bases 19 amino acid sequence of entire PRG4-LUB: 4 protein: 1069 amino acids 20 Lub: 4 DNA insert from cDNA cassette-1 and three synthetic cDNA cassette-2 sequences: 421 bases 21 139 amino acids encoded by Lub: 4 DNA insert (12 KEPAPTT sequences between S373 and E513 in SEQ ID NO: 19) 22 recombinant PRG4-Lub: 5 cDNA construct: 3303 bases 23 amino acid sequence of entire PRG4-LUB: 5 protein: 1100 amino acids 24 Lub: 5 DNA insert from cDNA cassette-1 and four synthetic cDNA cassette-2 sequences: 514 bases 25 170 amino acids encoded by Lub: 5 DNA insert (15 KEPAPTT sequences between S373 and E544 in SEQ ID NO: 23) 26 amino acid sequence "APTTPKEPAPTTTKSAPTTPKEPAPTTT KEPAPTTPKEPAPTTTK" (45 amino acids) in preferred PRG4- LUB: N protein 27 amino acid sequence "KEPAPTTTKEPAPTTTKSAPTTP KEPAPTTP" (31 amino acids) repeated N-1 times in preferred PRG4-LUB: N protein 28 amino acid sequence "EPAPTTTKSAPTTPKEPAPTTP" (22 amino acids) joining SEQ ID NO: 26 to (N-2) repeats of SEQ ID NO: 27 in preferred PRG4-LUB: N protein where N .gtoreq. 3. 29 amino acid sequence "KEPKPAPTTP" (10 amino acids) in preferred PRG4-LUB: N protein where N .gtoreq. 2.

[0012] The invention also provides in related embodiments a composition comprising a therapeutically effective amount of a recombinant lubricin protein in a pharmaceutically acceptable carrier. In some embodiments, the composition additionally comprises hyaluronan or hylan.

[0013] The invention further provides a method of treating a subject comprising: obtaining a recombinant lubricin protein composition; and administering said composition to a tissue of the subject. In related embodiments of this method of the invention, the tissue is selected from the group consisting of cartilage, synovium, meniscus, tendon, peritoneum, pericardium, and pleura. In further related embodiments of this method of the invention, the method additionally comprises a step selected from the group consisting of: providing an anesthetic to the subject; providing an anti-inflammatory drug to the subject; providing an antibiotic to the subject; aspirating fluid from the subject; washing tissue of the subject; and imaging tissue of the subject. In other related embodiments, the subject is selected from the group consisting of a mouse, a rat, a cat, a dog, a horse, and a human.

[0014] In other embodiments, the invention also provides an expression vector comprising a polynucleotide encoding a recombinant lubricin protein wherein the polynucleotide is operably linked to an expression control sequence. In related embodiments, the invention provides a method of producing recombinant lubricin protein comprising: growing cells transformed with the expression vector in liquid culture media; and collecting recombinant lubricin protein from the media. The collecting protein step may further comprise: concentrating the protein by filtering the media through a membrane; collecting the retained protein from the membrane; and solubilizing the collected protein in a buffered salt solution containing L-arginine hydrochloride ranging in concentration from 0.1 to 2.0 M.

[0015] In another related embodiment, the invention provides isolated antibody specific for a recombinant lubricin protein.

[0016] Other features and advantages of the invention will be apparent from the following description of preferred embodiments thereof, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The base DNA construct utilized in generating recombinant lubricin proteins may include variable arrangements of sequences 5' and 3' of exon 6 of the PRG4 gene. For example, the base DNA construct may include variable arrangements of sequences encoding somatomedin B-like domains (exons 2 through 4) or hemopexin-like domains (exons 7 through 9).

[0018] Embodiments of the base DNA construct having various exon arrangements 3' of exon 6 may include base DNA constructs that include only exon 7, 8, 9, 10, 11, or 12 individually, or exon pairs (7 and 8), (7 and 9), (7 and 10), (7 and 11), (7 and 12), (8 and 9), (8 and 10), (8 and 11), (8 and 12), (9 and 10), (9 and 11), (9 and 12), (10 and 11), (10 and 12), or (11 and 12), or exon triplets (7, 8 and 9), (7, 8 and 10), (7, 8, and 11), (7, 8, and 12), (7, 9 and 10), (7, 9 and 11), (7, 9 and 12), (7, 10 and 11), (7, 10 and 12), (7, 11 and 12), (8, 9 and 10), (8, 9 and 11), (8, 9 and 12), (8, 10 and 11), (8, 10 and 12), (8, 11 and 12), (9, 10 and 11), (9, 10 and 12), (9, 11 and 12), or (10, 11 and 12), or exon quadruplets (7, 8, 9 and 10), (7, 8, 9 and 11), (7, 8, 9 and 12), (7, 8, 10 and 11), (7, 8, 10 and 12), (7, 8, 11 and 12), (7, 9, 10 and 11), (7, 9, 10 and 12), (7, 9, 11 and 12), (7, 10, 11 and 12), (8, 9, 10 and 11), (8, 9, 10 and 12), (8, 9, 11 and 12), (8, 10, 11 and 12), or (9, 10, 11 and 12), or exon quintets (7, 8, 9, 10 and 11), (7, 8, 9, 10 and 12), (7, 8, 9, 11 and 12), (7, 8, 10, 11 and 12), (7, 9, 10, 11 and 12), or (8, 9, 10, 11 and 12), or exon sextet (7, 8, 9, 10, 11 and 12).

[0019] In addition, embodiments of the base DNA construct having various exon arrangements 5' of exon 6 may include base DNA constructs that include only exon 1, 2, 3, 4, or 5 individually, or exon pairs (1 and 2), (1 and 3), (1 and 4), (1 and 5), (2 and 3), (2 and 4), (2 and 5), (3 and 4), (3 and 5), or (4 and 5), or exon triplets (1, 2 and 3), (1, 2 and 4), (1, 2 and 5), (1, 3 and 4), (1, 3 and 5), (1, 4 and 5), (2, 3 and 4), (2, 3 and 5), (2, 4 and 5), or (3, 4 and 5), or exon quadruplets (1, 2, 3 and 4), (1, 2, 3 and 5), (1, 2, 4 and 5), (1, 3, 4 and 5), or (2, 3, 4 and 5), or exon quintets (1, 2, 3, 4 and 5).

[0020] The present invention also encompasses proteins encoded by base DNA constructs, i.e., wherein part or all of exon 6 sequence-encoded polypeptide is deleted and no amino acids encoded by inserts from synthetic cDNA cassettes have been added.

[0021] The present invention also encompasses polynucleotides that are homologous to the specific embodiments outlined herein, e.g., having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to the specified DNA sequences. The invention further includes polynucleotides having nucleic acid sequence capable of hybridizing over the length of a functional domain to the complement of the specified DNA sequences under high stringency conditions. The invention also includes proteins encoded by these homologous or hybridizing polynucleotides.

[0022] In order to delineate more clearly embodiments of the present invention, the following definitions are provided.

[0023] Definitions. The phrase "repetitive KEPAPTT-like sequence" means an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or higher identity to: (a) sequence "APTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTK" (SEQ ID NO: 26; 45 amino acids) and having at least one O-linked substitution; (b) sequence "KEPAPTTTKEPAPTTTKSAPTTPKEPAPTTP" (SEQ ID NO: 27; 31 amino acids) and having at least one O-linked substitution; or (c) sequence "EPAPTTTKSAPTTPKEPAPTTP" (SEQ ID NO: 28; 22 amino acids) and having at least one O-linked substitution. A repetitive KEPAPTT-like sequence may preferably have two, three, four or more O-linked substitutions.

[0024] While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans and has a definite meaning with respect to a given specified method. Sequence identity described herein is measured using the BLAST 2 SEQUENCES tool available through NCBI (http://www.ncbi.nlm.nih.gov/blast/; see also Tatusova and Madden (1999)). For amino acid sequences, the parameters used are expect=1000; word size=2; filter=off; and other parameters set to default values. These same parameters are used for nucleic acid sequences, except word size=8. Default values for amino acid sequence comparisons are: Matrix=BLOSUM62; open gap=11; extension gap=1 penalties; and gap.times.dropoff=50. Default values for nucleic acid sequence comparisons are: reward for a match=1; penalty for a mismatch=-2; strand option=both strands; open gap=5; extension gap=2 penalties; and gap.times.dropoff=50.

[0025] An O-linked substitution of recombinant lubricin may be a substitution with the lubricating oligosaccharide .beta.-(1-3)-Gal-GalNac, or with other moieties, including artificial or naturally-occurring carbohydrate moieties (such as keratan sulfate or chondroitin sulfate). In some embodiments, the O-linked substitution may be with moieties that contribute to a capacity of recombinant lubricin to act as a carrier of surface active phospholipid (SAPL) or surfactants (Hills, 2002). Percent glycosylation or substitution is determined by weight (dry weight).

[0026] High stringency conditions, when used in reference to DNA:DNA hybridization, comprise conditions equivalent to binding or hybridization at 42.degree. C. in a solution consisting of 5.times. SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4.H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5.times. Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1.times. SSPE, 1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in length is employed.

[0027] Polypeptides or other compounds described herein are said to be "isolated" when they are within preparations that are at least 50% by weight (dry weight) the compound of interest. Polypeptides or other compounds described herein are said to be "substantially pure" when they are within preparations that are at least 80% by weight (dry weight) the compound of interest. Polypeptides or other compounds described herein are said to be "homogeneous" when they are within preparations that are at least 95%, and preferably 99%, by weight (dry weight) the compound of interest. Purity is measured by reducing polyacrylamide gel electrophoresis and enhanced coomassie blue staining, followed by optical density traces of bands (i.e., with protein purity being measured through optical densitometry).

[0028] "Pyrogen-free" means free of fever causing contaminants, including endotoxin. Measurement of contaminants is to be performed by the applicable standard tests set by the U.S. Food and Drug Administration.

[0029] As used herein, the term "therapeutically effective amount" means the total amount of each active component of the relevant pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0030] Embodiments of the present invention may be used as intra-articular supplements. Intra-articular supplementation with compounds not derived from lubricin has been practiced as a joint therapy. For example, "viscosupplementation" with polymeric hyaluronan (HA) and higher molecular weight hylans (such as SYNVISC.RTM. elastoviscous fluid "Hylan G-F 20"-distributed by WYETH.RTM. Pharmaceuticals) is used clinically to treat OA-associated knee pain. This viscosupplementation has shown significant therapeutic value, particularly in reducing weight-bearing pain in patients (Wobig et al., 1998).

[0031] Hylan G-F 20 is generated by cross-linking several HA molecules obtained from rooster or chicken combs. Viscosupplementation with Hylan G-F 20 can be significantly more efficacious for alleviating pain than viscosupplementation with lower molecular weight HA (Wobig et al., 1999). In addition, relieving pain by viscosupplementation with Hylan G-F 20 may be particularly preferable to administration of NSAIDs for those patients who do not tolerate NSAIDs (e.g., in patients with a high risk of gastrointestinal complications; Espallargues and Pons, 2003). Though Hylan G-F 20 viscosupplementation is a safe and well-tolerated therapy that provides a short-term (i.e., until 3-6 months posttreatment) decrease in pair symptoms while improving joint function, the therapy may not significantly forestall the eventual need for knee replacement in OA patients (Espallargues and Pons, 2003).

EXAMPLE 1

Cloning of Recombinant Lubricin

[0032] Constructs. In some embodiments, the base DNA construct for the generation of recombinant lubricin molecules is composed of the Met codon (ATG) through the BssHII restriction site (G CGCGC) of SEQ ID NO: 6 (i.e., base nos. 1 through 1123) and the BspEI restriction site (T CCGGA) through the stop codon (TAA) of SEQ ID NO: 6 (i.e., base nos. 1269 through 2946). These sequences, i.e., base nos. 1 through 1123 and 1269 through 2946 of SEQ ID NO: 6, encode amino acids M1 through 5373 (encoded by exons 1 through 5 and approximately 174 flanking 5'-codons of exon 6) and E848 through P1404 (encoded by approximately 293 flanking 3'-codons of exon 6 and exons 7 through 14) of native full-length lubricin (i.e., PRG4). The portion of exon 6 absent from the base DNA construct corresponds to DNA sequence encoding amino acids A374 through P847 of native PRG4 (474 amino acids absent out of approximately 940 amino acids encoded by exon 6). This absent amino acid sequence is rich in KEPAPTT-like sequences.

[0033] DNA sequence of synthetic cDNA cassette-1 (SEQ ID NO: 1) is added BssHII/BspEI to the base construct to make the recombinant PRG4-Lub:1 cDNA construct (SEQ ID NO: 6). SEQ ID NO: 6 is composed of the Lub:1 DNA insert (SEQ ID NO: 8; which encodes the 51 amino acids of SEQ ID NO: 9 with its four KEPAPTT sequences) between DNA encoding amino acids M1 through 5373 and DNA encoding E848 through P1404 of native PRG4. In other words, in place of A374 through P847 (474 amino acids) of native PRG4, the recombinant lubricin PRG4-LUB:1 includes 51 amino acids that form four perfect KEPAPTT sequences and approximately three imperfect KEPAPTT sequences.

[0034] DNA sequence of synthetic cDNA cassette-2 (SEQ ID NO: 3) is added Bsu36I/BspEI to the PRG4-Lub:1 construct to make the PRG4-Lub:2 cDNA construct (SEQ ID NO: 10). The PRG4-Lub:1 cDNA construct has one Bsu36I restriction site (CC TNAGG, i.e., CC TAAGG; base nos. 1225 through 1231 of SEQ ID NO: 6). When synthetic cDNA cassette-2 is added to the PRG4-Lub:1 cDNA construct, this Bsu36I site is destroyed, but synthetic cassette-2 contains another internal Bsu36I restriction site (CC TNAGG, i.e., CC TAAGG; base nos. 92 through 98 of SEQ ID NO: 3). Consequently, a PRG4-Lub:N+1 construct can be made by adding synthetic cDNA cassette-2 Bsu36I/BspEI to the previous PRG4-Lub:N construct at this internal Bsu36I restriction site provided by synthetic cDNA cassette-2.

[0035] The cDNA cassettes are synthesized as single stranded oligonucleotides and hybridized together to produce a double stranded DNA fragment with sticky ends. This is why the terminal BssHII, Bsu36I, and BspEI sites appear incomplete. In synthetic cDNA cassette-1 (SEQ ID NO: 1), a sequence bounded by remnant flanking BssHII (G CGCGC) and BspEI (T CCGGA) restriction sites includes an internal Bsu36I restriction site (CC TNAGG, i.e., CC TAAGG); the restriction sites are underlined below:

TABLE-US-00002 CGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCT CCTACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCAC CCACCACGCCTAAGGAGCCAGCTCCTACTACAACGAAACCGGCACCAAC CACTCCGG

[0036] SEQ ID NO: 2, which is a translation of SEQ ID NO: 1, includes four KEPAPTT sequences that are perfect matches (highlighted below):

TABLE-US-00003 1 A P T T P K E P A P T T T K S A P T T P CGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCTACTACGCCC 21 K E P A P T T T K E P A P T T P K E P A AAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCACGCCTAAGGAGCCAGCT 41 P T T T K P A P T T P CCTACTACAACGAAACCGGCACCAACCACTCCGG

[0037] Synthetic cDNA cassette-2 (SEQ ID NO: 3) similarly has a remnant 5'-terminal Bsu36I restriction site (i.e., CC TNAGG, evidenced only by the TAA sequence), a 3'-terminal remnant BspEI restriction site (T CCGGA), and an internal Bsu36I restriction site (CC TNAGG); the restriction sites are underlined below:

TABLE-US-00004 TAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGA AGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAGGA ACCCAAACCGGCACCAACCACTCCGG

[0038] SEQ ID NO: 4, which is a translation of SEQ ID NO: 3, includes three KEPAPTT sequences that are perfect matches (highlighted below):

TABLE-US-00005 1 K E P A P T T T K E P A P T T T K S A P TAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCC 21 T T P K E P A P T T P K E P K P A P T T ACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAGGAACCCAAACCGGCACCAACCACT 41 P CCGG

[0039] The recombinant PRG4-Lub:1 cDNA construct (SEQ ID NO: 6) in pTmed2 vector (construct plus vector equals SEQ ID NO: 5) is flanked by SalI (G TCGAC; base nos. 1027 through 1032 of SEQ ID NO: 5) and NotI (GC GGCCGC; base nos. 3984 through 3991 of SEQ ID NO: 5) restriction sites. The SalI site incorporates a modified Kozak translation initiation sequence (CCCACC; base nos. 1032 through 1037 of SEQ ID NO: 5) before the translation start codon ATG (base nos. 1038 through 1040 of SEQ ID NO: 5). Between the BssHII (G CGCGC; base nos. 2155 through 2160 of SEQ ID NO: 5) and BspEI (T CCGGA; base nos. 2306 through 2311 of SEQ ID NO: 5) restriction sites is found the internal Bsu36I cloning site (CC TNAGG, i.e., CC TAAGG; base nos. 2262 through 2268 of SEQ ID NO: 5).

[0040] The PRG4-Lub:1 cDNA construct (SEQ ID NO: 6) is translated into the PRG4-LUB:1 protein (SEQ ID NO: 7). The insert between 5373 and E425 (i.e., E848 of native PRG4) of the entire PRG4-LUB:1 protein (SEQ ID NO: 7) is the 51 amino acids of SEQ ID NO: 9. These are translated from the Lub:1 DNA insert (SEQ ID NO: 8) and include four perfect KEPAPTT sequences. Between the BssHII restriction site (G CGCGC; base nos. 1118 through 1123 of SEQ ID NO: 6) and the BspEI restriction site (T CCGGA; base nos. 1269 through 1274 of SEQ ID NO: 6) is found the internal Bsu36I cloning site (CC TNAGG, i.e., CC TAAGG; base nos. 1225 through 1231 of SEQ ID NO: 6).

[0041] As in the recombinant PRG4-Lub:1 construct in pTmed2 vector, the recombinant PRG4-Lub:2 cDNA construct (SEQ ID NO: 10) in pTmed2 vector is flanked by SalI (G TCGAC) and NotI (GC GGCCGC) restriction sites; the SalI site incorporates a modified Kozak translation initiation sequence (CCCACC) before the translation start codon ATG (base nos. 1 through 3 of SEQ ID NO: 10). Similarly, the recombinant PRG4-Lub:3 cDNA construct (SEQ ID NO: 14), the recombinant PRG4-Lub:4 cDNA construct (SEQ ID NO: 18), and the recombinant PRG4-Lub:5 cDNA construct (SEQ ID NO: 22) in pTmed2 vector are each flanked by SalI (G TCGAC) and NotI (GC GGCCGC) restriction sites; the SalI site incorporates a modified Kozak translation initiation sequence (CCCACC) before the translation start codon ATG (base nos. 1 through 3 of SEQ ID NOS: 14, 18, and 22, respectively).

[0042] Within the PRG4-Lub:2 cDNA construct, the internal Bsu36I cloning site (CC TNAGG, i.e., CC TAAGG; base nos. 1318 through 1324 of SEQ ID NO: 10) is found between the BssHII (G CGCGC; base nos. 1118 through 1123) and BspEI (T CCGGA; base nos. 1347 through 1352) restriction sites. The PRG4-Lub:2 construct (SEQ ID NO: 10) is translated into the PRG4-LUB:2 protein (SEQ ID NO: 11). The insert between S373 and E451 (i.e., E848 of native PRG4) of the entire PRG4-LUB:2 protein (SEQ ID NO: 11) is the 77 amino acids of SEQ ID NO: 13. These are translated from the Lub:2 DNA insert (SEQ ID NO:12). In place of A374 through P847 (474 amino acids) of native PRG4, the 77 amino acids of the recombinant lubricin PRG4-LUB:2 form six perfect KEPAPTT sequences and approximately four imperfect KEPAPTT sequences.

[0043] Within the PRG4-Lub:3 cDNA construct, the internal Bsu36I cloning site (CC TNAGG, i.e., CC TAAGG; base nos. 1411 through 1417 of SEQ ID NO: 14) is found between BssHII (G CGCGC; base nos. 1118 through 1123) and BspEI (T CCGGA; base nos. 1440 through 1445) restriction sites. The PRG4-Lub:3 construct (SEQ ID NO: 14) is translated into the PRG4-LUB:3 protein (SEQ ID NO: 15). The insert between S373 and E482 (i.e., E848 of native PRG4) of the entire PRG4-LUB:3 protein (SEQ ID NO: 15) is the 108 amino acids of SEQ ID NO: 17. These are translated from the Lub:3 DNA insert (SEQ ID NO:16). In place of A374 through P847 (474 amino acids) of native PRG4, the 108 amino acids of the recombinant lubricin PRG4-LUB:3 form nine perfect KEPAPTT sequences and approximately five imperfect KEPAPTT sequences.

[0044] Within the PRG4-Lub:4 cDNA construct, the internal Bsu36I cloning site (CC TNAGG, i.e., CC TAAGG; base nos. 1504 through 1510 of SEQ ID NO: 18) is found between BssHII (G CGCGC; base nos. 1118 through 1123) and BspEI (T CCGGA; base nos. 1533 through 1538) restriction sites. The PRG4-Lub:4 construct (SEQ ID NO: 18) is translated into the PRG4-LUB:4 protein (SEQ ID NO: 19). The insert between S373 and E513 (i.e., E848 of native PRG4) of the entire PRG4-LUB:4 protein (SEQ ID NO: 19) is the 139 amino acids of SEQ ID NO: 21. These are translated from the Lub:4 DNA insert (SEQ ID NO:20). In place of A374 through P847 (474 amino acids) of native PRG4, the 139 amino acids of the recombinant lubricin PRG4-LUB:4 form twelve perfect KEPAPTT sequences and approximately six imperfect KEPAPTT sequences.

[0045] Within the PRG4-Lub:5 cDNA construct, the internal Bsu36I cloning site (CC TNAGG, i.e., CC TAAGG; base nos. 1597 through 1603 of SEQ ID NO: 22) is found between BssHII (G CGCGC; base nos. 1118 through 1123) and BspEI (T CCGGA; base nos. 1626 through 1631) restriction sites. The PRG4-Lub:5 construct (SEQ ID NO: 22) is translated into the PRG4-LUB:5 protein (SEQ ID NO: 23). The insert between S373 and E544 (i.e., E848 of native PRG4) of the entire PRG4-LUB:5 protein (SEQ ID NO: 23) is the 170 amino acids of SEQ ID NO: 25. These are translated from the Lub:5 DNA insert (SEQ ID NO:24). In place of A374 through P847 (474 amino acids) of native PRG4, the 170 amino acids of the recombinant lubricin PRG4-LUB:5 form fifteen perfect KEPAPTT sequences and approximately seven imperfect KEPAPTT sequences.

[0046] Importantly, the process of inserting the synthetic cDNA cassette-2. can be iterated indefinitely. Each iteration results in the addition of three perfect KEPAPTT sequences. Just as recombinant lubricins PRG4-LUB:2 through PRG4-LUB:5 are constructed in this way through the use of insert sequences, recombinant lubricins PRG4-LUB:6 through PRG4-LUB:N are constructed. Table 2 provides a summary of BssHII/BspEI insert sequences.

TABLE-US-00006 TABLE 2 BssHII|BspE1 Insert Sequences LUB SEQ Sequences (restriction sites underlined in DNA inserts; INSERT ID NO: KEPAPTT sequences are highlighted in protein inserts) Lub: 1 8 GCGCGCCCACAACTCCAAAAGAGGCCGCACCTACCACGACAAAGTCAGCTCCT ACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCAC GCCTAAGGAGCCAGCTCCTACTACAACGAAACCGGCACCAACCACTCCGGA LUB: 1 9 APTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTKPAPTTP Lub: 2 12 GCGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCT ACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCAC GCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGA AGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAGGAACCC AAACCGGCACCAACCACTCCGGA LUB: 2 13 APTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTKEPAPTTTK SAPTTPKEPAPTTPKEPKPAPTTP Lub: 3 16 GCGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCT ACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCAC GCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGA AGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCA GCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCAC AACACCAAAGGAGCCGGCCCCTACGACTCCTAAGGAACCCAAACCGGCACCAA CCACTCCGGA LUB: 3 17 APTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTKEPAPTTTK SAPTTPKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKEPAPTTPKEPKPAPT TP Lub: 4 20 GCGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCT ACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCAC GCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGA AGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCA GCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCAC AACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCAGCCCCTACTACGA CAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAG CCGGCCCCTACGACTCCTAAGGAACCCAAACCGGCACCAACCACTCCGGA LUB: 4 21 APTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTKEPAPTTTK SAPTTPKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKEPAPTTPKEPAPTTT KEPAPTTTKSAPTTPKEPAPTTPKEPKPAPTTP Lub: 5 24 GCGCGCCCACAACTCCAAAAGAGCCCGCACCTACCACGACAAAGTCAGCTCCT ACTACGCCCAAAGAGCCAGCGCCGACGACTACTAAAGAACCGGCACCCACCAC GCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGA AGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCA GCCCCTACTACGACAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCAC AACACCAAAGGAGCCGGCCCCTACGACTCCTAAAGAACCAGCCCCTACTACGA CAAAGGAGCCTGCACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAG CCGGCCCCTACGACTCCTAAAGAACCAGCCCCTACTACGACAAAGGAGCCTGC ACCCACAACCACGAAGAGCGCACCCACAACACCAAAGGAGCCGGCCCCTACGA CTCCTAAGGAACCCAAACCGGCACCAACCACTCCGGA LUB: 5 25 APTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTKEPAPTTTK SAPTTPKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKEPAPTTPKEPAPTTT KEPAPTTTKSAPTTPKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKEPAPTT PKEPKPAPTTP

[0047] Although we have exemplified the base DNA construct with full-length PRG4 containing all 12 exons (minus a central portion of exon 6), splice variants of PRG4 may also be employed, depending on the various activities and length desired. Additionally, different restrictions enzymes may be employed in an analogous strategy, providing that their location is conveniently located within nucleic acid sequence encoding PRG4 protein. In other embodiments, the base DNA construct lacks native exon 6 sequence, but includes one or more of exon 1 through exon 5 sequences or of exon 7 through exon 12 sequences of the native PRG4 gene. In other embodiments, the base DNA construct is identical to a recombinant MSF sequences described in U.S. Pat. No. 6,433,142 or US20020137894 except that part or all of the sequences of exon 6 are absent.

[0048] The invention provides cDNA constructs encoding recombinant lubricins that are cloned into SalI (G TCGAC; base nos. 1027 through 1032 of SEQ ID NO: 5) and NotI (GC GGCCGC; base nos. 3984 through 3991 of SEQ ID NO: 5) restriction sites in the eucaryotic expression vector pTmed2 as a preferred embodiment (e.g., recombinant PRG4-Lub:1 cDNA construct in pTmed2 expression vector is located in SEQ ID NO: 5 at base nos. 1038 though 3983). The SalI site incorporates the first base of a modified Kozak translation initiation sequence (CCCACC; base no. 1032 of SEQ ID NO: 5) before the methionine start codon (ATG; base nos. 1038 through 1040 of SEQ ID NO: 5). Other embodiments of the invention include other restriction site combinations and other expression vectors.

[0049] In a preferred embodiment, the interative process makes use of the synthetic cDNA cassette-1 (SEQ ID NO: 1) in expression vector pTmed2, which is flanked by the restriction sites for BssHII (G CGCGC) and BspEI (T CCGGA), and the synthetic cDNA cassette-1, which includes an internal Bsu36I restriction site (CC TNAGG, i.e., CC TAAGG; base nos. 107 to 113 of SEQ ID NO: 1). For the iterative generation of recombinant lubricin constructs containing KEPAPTT-like sequences in this preferred embodiment, synthetic cDNA cassette-2 (SEQ ID NO: 3) is inserted between the Bsu36I and BspEI sites of the recombinant construct. Synthetic cDNA cassette-2 (SEQ ID NO: 3) is flanked by a modified remnant Bsu36I site (TAAAG) and a remnant BspEI (ACTCCGG) site. It also includes an internal Bsu36I site (CC TNAGG, i.e., CC TAAGG; base nos. 92 through 98 of SEQ ID NO: 3). Upon cloning synthetic cDNA cassette-2 into the Bsu36I and BspEI sites of a recombinant lubricin construct, the Bsu36I cloning site of the original construct is destroyed leaving one unique Bsu36I cloning site in the new construct.

[0050] In this preferred embodiment, the amino acid sequence "APTTPKEPAPTT TKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTK" (SEQ ID NO: 26; 45 amino acids) remains a part of each PRG4-LUB:N protein (where N=an integer of 1 or more). In addition, the amino acid sequence "KEPAPTTTKEPAPTTTKSAPTTPKEPAPTTP" (SEQ ID NO: 27; 31 amino acids) is encoded by the DNA insert that becomes part of each PRG4-Lub:N+1 cDNA construct through the addition of synthetic cDNA cassette-2 Bsu36I/BspEI to a PRG4-Lub:N cDNA construct. For PRG4-LUB:N protein where N is an integer greater than or equal to 3, the amino acid sequence "EPAPTTTKSAPTTPKEPAPTTP" (SEQ ID NO: 28; 22 amino acids) joins SEQ ID NO: 26 to (N minus 2) repeats of SEQ ID NO: 27 in preferred embodiments. Furthermore, the amino acid sequence "KEPKPAPTTP" (SEQ ID NO: 29; 10 amino acids) immediately follows the last insert repeat of SEQ ID NO: 27 in preferred embodiments of the PRG4-LUB:N protein where N is an integer greater than or equal to 2.

[0051] Because they form at least two KEPAPTT sequences, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 are each designated herein to be a "repetitive KEPAPTT-like sequence" (the N-terminus of SEQ ID 28 links to a K residue so that SEQ ID NO: 28 forms two KEPAPTT sequences in PRG4-LUB:N proteins).

[0052] Consequently, for recombinant lubricin protein. PRG4-LUB:N (where N equals an integer of 1 or more), the PRG4-LUB:N protein comprises SEQ ID NO: 26 in a preferred embodiment. Furthermore, for recombinant lubricin protein PRG4-LUB:N (where N equals an integer of 2 or more), the PRG4-LUB:N protein also comprises SEQ ID NO: 27 in a preferred embodiment. SEQ ID NO: 27 is repeated (N minus 1) times within each PRG4-LUB:N protein in these preferred embodiments. In PRG4-LUB:2, SEQ ID NO: 26 and SEQ ID NO: 27 overlap (i.e., they share a KEPAPTT sequence).

[0053] In other preferred embodiments where N is an integer greater than or equal to 3 (e.g., where N equals an integer from 3 through 200, or in more preferred embodiments where N equals an integer from 5 through 50, or in even more preferred embodiments where N equals an integer from 10 through 30), recombinant lubricin protein comprises the 22 amino acids of SEQ ID NO: 28 joining the N-terminal-oriented 45 amino acids of SEQ ID NO: 26 to (N minus 2) repeat(s) of the 31 amino acids of SEQ ID NO: 27, where the 10 amino acids of SEQ ID NO: 29 are C-terminal to the last 31-amino-acid repeat of SEQ ID NO: 27.

TABLE-US-00007 TABLE 3 Sequence Frequencies in Preferred PRG4-LUB Proteins SEQ ID SEQ ID SEQ ID SEQ ID PRG4-LUB NO: 26 NO: 28 NO: 27 NO: 29 KEPAPTT Protein N-end insert >--< >--< insert C-end repeats -LUB:1 1 0 0 0 4 -LUB:2 1 0 1 1 6 -LUB:3 1 1 1 1 9 -LUB:4 1 1 2 1 12 -LUB:5 1 1 3 1 15 -LUB:N 1 1 N - 2 1 3 .times. N

[0054] PRG4-LUB:N proteins in general have (3 times N) repeats of the KEPAPTT sequence in preferred embodiments where N equals the number of repetitive KEPAPTT-like sequences. Recombinant lubricin PRG4-LUB:5 (having 3.times.N=3.times.5=15 copies of the KEPAPTT sequence in preferred embodiments) is the largest recombinant lubricin PRG4-LUB:N whose sequence is detailed herein. For recombinant lubricin of the present invention, however, the value N may be greater than 5, such as 7, 10, 12, 15, 20, 25, 30, 40, 50, 100, 150, 200 or more.

[0055] In particular, proteins PRG4-LUB:1, PRG4-LUB:2, PRG4-LUB:3, PRG4-LUB:4, and PRG4-LUB:5 are detailed herein with 4, 6, 9, 12 and 15 perfect KEPAPTT sequences, respectively. However, it is possible to add increasing numbers of KEPAPTT sequences by continuing the iterative Lub:N insert procedure described herein. We have provided detailed description for PRG4-LUB:N recombinant lubricins with relatively low numbers of KEPAPTT or KEPAPTT-like sequences as compared with native PRG4/lubricin protein because smaller proteins are easier to synthesize and manipulate.

[0056] It may also be desirable to increase the number of KEPAPTT-like sequences over that seen in native PRG4 protein. This can be accomplished either by continuing the iterative Lub:N insert procedure described herein so that there are more than 78 KEPAPTT-like sequences in the recombinant lubricin PRG4-LUB:N protein, or by beginning with an intact PRG4 cDNA, rather than an exon 6-deleted or an exon 6-diminished version of PRG4 cDNA. Thus any KEPAPTT-like sequences added will be in excess of the number found in native PRG4 protein. Insert procedures used for the generation of larger recombinant lubricin proteins from an intact PRG4 cDNA, as well as insert procedures that use an exon 6-deleted or an exon 6-diminished version of PRG4 cDNA, are encompassed within the invention.

EXAMPLE 2

Expression and Purification of `Lub` Protein

[0057] PRG4-Lub:1 cDNA construct (SEQ ID NO: 6; containing synthetic cDNA cassette-1 sequence) was expressed in a stably transfected, preadaptive CHO DUKX cell line, purified from conditioned media, and solubilized in PBS containing 500 mM L-arginine hydrochloride as follows.

[0058] The PRG4-Lub:1 cDNA construct was expressed in a stably transfected CHO DUKX cell line and the conditioned media was collected. A two liter volume of this conditioned media was filter concentrated under compressed nitrogen gas (40 psi) using an AMICON.RTM. M2000.TM. filtration unit fitted with either a 10 kDa nominal molecular weight limit (NMWL), a 30 kDa NMWL or a 100 kDa NMWL PALL FILTRON.RTM. OMEGA.TM. disc membrane. Media was concentrated to approximately a 100 ml volume, which was aspirated from the disc membrane. The disc membrane was then removed from the AMICON.RTM. M2000.TM. filtration unit. The "mucinous" retentate, which had accumulated at the surface of the disc membrane, was harvested using a cell scraper and transferred to microcentrifuge tubes. The samples in the microcentrifuge tubes were centrifuged at approximately 12,000.times.g for 10 minutes, and the aqueous supernatant was removed. The remaining "lubricin-enriched" pellets were dissolved in phosphate buffered saline (PBS) containing 500 mM L-arginine hydrochloride. The L-arginine hydrochloride concentration may range from 100 mM to 2.0 M.

[0059] Using the above procedure, PRG4-LUB:2 through PRG4-LUB:5 glycoproteins (and PRG4-LUB:N proteins where N=a nonnegative integer of 6 or more, as well as other glycoproteins containing KEPAPTT-like sequences) are harvested directly from disc membranes, i.e., without purification of the concentrate remaining above disc membranes. That is, these recombinant lubricin glycoproteins are isolated directly from disc membranes of 10 kDa NMWL, 30 kDa NMWL, or 100 kDa NMWL PALL FILTRON.RTM. OMEGA.TM. filtration units. In some instances, these glycoproteins may also be purified from the concentrate remaining above disc membranes through chromatographic techniques or electrophoretic techniques or both. Recombinant lubricin proteins and glycoproteins may also be purified using chromatography and other techniques known in the art (as, for example, described in U.S. Pat. No. 6,433,142 for MSF proteins; see also: Deutscher, 1990; and Scopes, 1994).

EXAMPLE 3

Immunohistochemistry

[0060] The cell source of lubricin in normal and osteoarthritic joints was further investigated using immunohistochemical techniques. In addition, the presence of lubricin on other tissue surfaces, including pleura, pericardium, peritoneum, and meninges, was examined according to the following methods.

[0061] Osteoarthritic cartilage and synovium were obtained by informed consent from patients undergoing knee replacement surgery. Other tissues examined were normal human synovium and normal non-human primate (NHP) synovium, cartilage, pleura, pericardium, peritoneum, meninges, brain, tendon, and ligaments, and canine normal and osteoarthritic meniscus, cartilage, synovium, ligament, and tendons. Tissues were fixed in 4% paraformaldehyde immediately after harvest or following 24 hours incubation in media without and with supplemental monensin (5 .mu.M). For immunohistochemical studies the tissues were fixed in 4% paraformaldehyde for 24 hours and 6-8 micron paraffin sections were obtained. A subset of tissues were frozen in optical coherence tomography (OCT) freezing compound and cut at 5 to 10 micron intervals followed by acetone fixation.

[0062] Immunohistochemical and immunofluorescent analyses utilized a purified polyclonal rabbit anti-human lubricin antibody (Ab 06A10) generated by immunization with a truncated form of recombinant lubricin and purification on a protein A column. CD16 antibody (NEOMARKERS.RTM., Fremont Calif.) was used to identify macrophages (Fcy receptor III). CD106/VCAM-1 antibody (NEOMARKERS.RTM.) was used to label fibroblasts within cryostat sections. For control sections, an equivalent concentration of RIgG (VECTOR LABS.TM., CA), MIgG.sub.1 (DAKO.RTM.), and MIgG.sub.2a (DAKO.RTM.) was used consecutively. The Dextran Technology System (ENVISION+.TM.; DAKO.RTM.) was used to visualize antibody binding and the sections were counterstained with Mayer's alum-hematoxylin. Immunofluorescence was performed using the above primary antibodies and probed with secondary antibodies (Alexa Dyes--MOLECULAR PROBES.TM., Oregon) goat anti-rabbit Alexa dye at 546 nm and goat anti-mouse Alexa dye at 488 nm. Fluorescent binding of the antibody was detected with a NIKON.RTM. fluorescent microscope.

[0063] Lubricin was detected along the surfaces of normal and osteoarthritic human articular cartilage and synovium. A thick layer of lubricin completely coated the fibrillated osteoarthritic surface. CD106 immunofluorescence showed strong cell membrane staining of the intimal fibroblasts of the synovium; lubricin protein was also visualized as staining within synovial cells. Double immunostaining for CD106+lubricin, clearly showed co-localization within the intimal fibroblasts of the synovium. CD16 staining of synovial macrophages demonstrated the presence of these cells throughout the layers of the synovium, but there was no co-localization with lubricin.

[0064] Staining of NHP and canine articular tissues (normal and OA) with the lubricin antibody showed lubricin coating the surface layer of the synovium, cartilage, meniscus, and tendons. NHP cartilage also showed strong immunoreactivity not only in the superficial zone cells but also the transitional zone cells without the addition of monensin to increase intracellular stores of the glycoprotein. Cells lining the peritoneum, pericardium, and pleura also exhibited lubricin expression, though no immunoreactivity was observed in the meninges or brain.

[0065] In summary, both normal and osteoarthritic synovium, tendon, meniscus and cartilage were coated by a substantial layer of lubricin. The glycoprotein is clearly present on tissues within OA joints. Double-immunofluorescent staining of human OA synovium demonstrated that the intimal fibroblast synoviocytes were responsible for the synthesis of lubricin.

[0066] The localization of lubricin protein outside joint tissue has not been previously described. A surface layer of lubricin was clearly demonstrated on lung pleura, pericardium, and peritoneum. Lubricin is reputed to have a lubricating function within the synovial joint, but may have multiple roles including, but not limited to, lubrication and anti-adhesive functions in other tissues. Supplementation of these other tissues with lubricin is a biotherapy encompassed within this invention.

EXAMPLE 4

Recombinant Lubricin as a Mechanical Lubricant

[0067] Recombinant lubricin could be used as a lubricant generally, e.g., with seals and bearings and the like. For example, U.S. Pat. No. 3,973,781 entitled "Self-lubricating seal," U.S. Pat. No. 4,491,331 entitled "Grooved mechanical face seal," US4560174 entitled "Multi lip seal," and U.S. Pat. No. 4,973,068 entitled "Differential surface roughness dynamic seals and bearings," each describe seals of varying designs. Recombinant lubricin could be used as a lubricant with these seals.

[0068] In particular, recombinant lubricin could be used as a lubricant for medical devices, prostheses, and implants, particularly where a biocompatible lubricant is required. In addition, the applications need not be medical, but could include applications in environmentally sensitive contexts where a biocompatible lubricant may be desirable.

EXAMPLE 5

Recombinant Lubricin Compositions

[0069] A recombinant lubricin of the present invention may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or complement its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects.

[0070] Use of recombinant lubricin protein for intra-articular supplementation in combination with the previously described polymeric hyaluronan (HA) and higher molecular weight hylans is particularly preferred. Other preferred combinations for use in intra-articular supplementation include the use of recombinant lubricin protein with anesthetics (e.g., lidocaine), steroids (e.g., triamcinolone hexacetonide), or radioisotopes (e.g., yttrium). Other preferred combinations for use in intra-articular supplementation may include autologous or heterologous cell preparations (e.g., of cultured chondrocytes, synoviocytes, or stem cells, whether autologously or heterologously derived).

[0071] A recombinant lubricin of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.

[0072] A pharmaceutical composition of the invention may be in the form of a complex of the recombinant lubricin protein(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.

[0073] A pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871, U.S. Pat. No. 4,501,728, U.S. Pat. No. 4,837,028, and U.S. Pat. No. 4,737,323.

[0074] In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a subject (e.g., a mammal) having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines, other hematopoietic factors, or cell-based supplements. When co-administered with one or more cytokines, lymphokines, other hematopoietic factors, or cell-based supplements, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or cell-based supplement, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or cell-based supplement.

[0075] Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection, or, in some instances, oral ingestion, inhalation, topical application. Administration to a patient by injection into joint tissue is generally preferred (Schumacher, 2003).

[0076] When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.

[0077] When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For example, injection in association with, or in combination with, lidocaine or other local anesthetic, steroids or adrenocorticoids, HA and/or hylans, or radioisotopes are all encompassed within by the present invention.

[0078] The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 .mu.g to about 100 mg (preferably about 0.1 .mu.g to about 10 mg, more preferably about 0.1 .mu.g to about 1 mg) of protein of the present invention per kg body weight depending on the method of administration and the exact therapeutic course implemented.

[0079] If administered intravenously, the duration of intravenous therapy using a pharmaceutical composition comprising recombinant lubricin of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention may be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.

[0080] For compositions of the present invention which are useful for bone, cartilage, tendon or ligament therapy, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for in some wound healing and tissue repair contexts. Therapeutically useful agents which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition comprising recombinant lubricin protein of the invention in the methods of the invention. Preferably the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and/or cartilage damage, possibly capable of providing a structure for the developing bone and cartilage, and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.

[0081] If a matrix is used, the choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.

[0082] In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-.alpha. and TGF-.beta.), and insulin-like growth factor (IGF).

[0083] The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals such as cats and dogs, laboratory animals such as mice and rats, as well as horses, in addition to humans, are particularly desired subjects or patients for such treatment with recombinant lubricin proteins of the present invention.

[0084] The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., cartilage or tendon), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.

[0085] Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a subject (e.g., a mammal). Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).

[0086] Cells may also be cultured ex vivo in the presence of nucleic acids or proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

EXAMPLE 6

Anti-Lubricin Antibodies

[0087] Recombinant lubricin protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein or, in some embodiments, its native counterparts. Such antibodies may be obtained using either complete recombinant lubricin protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art (for example, as in Merrifield, 1963; and Krstenansky et al., 1987). Monoclonal antibodies binding to recombinant lubricin protein of the invention may be useful diagnostic agents for the immunodetection of related proteins. Neutralizing monoclonal antibodies binding to these related proteins may also be useful therapeutics for both conditions associated with lubricin or, in some cases, in the treatment of some forms of cancer where abnormal expression of lubricin may be involved (e.g., in synoviomas).

[0088] In addition to antibodies which are directed to the polypeptide core of a recombinant lubricin protein, an antibody directed to a sugar portion or to a glycoprotein complex of recombinant lubricin protein is desirable. In order to generate antibodies which bind to glycosylated recombinant lubricin (but not to a deglycosylated form), the immunogen is preferably a glycopeptide, the amino acid sequence of which spans a highly glycosylated portion of the recombinant lubricin, e.g., a repetitive KEPAPTT-like sequence. Shorter glycopeptides, e.g., 8-15 amino acids in length, within the same highly glycosylated region, are also used as immunogens. Methods of generating antibodies to highly glycosylated biomolecules are known in the art (for example, as described by Schneerson et al., 1980).

EXAMPLE 7

Recombinant Lubricin Delivery

[0089] Standard methods for delivery of recombinant lubricin are used. For intra-articular administration, recombinant lubricin is delivered to the synovial cavity at a concentration in the range of 20-500 .mu.g/ml in a volume of approximately 0.1-2 ml per injection. For example, 1 ml of a recombinant lubricin at a concentration of 200-300 .mu.g/ml is injected into a knee joint using a fine (e.g., 14-30 gauge, preferably 18-26 gauge) needle. The compositions of the invention are also useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, or intraperitoneal administration, and, in preferred embodiments, onto the surfaces of the peritoneal, pericardium, or pleura.

[0090] Proper needle placement is critical for the efficacy of recombinant lubricin protein that is delivered by injection in joint therapies (Schumacher, 2003). Proper needle placement may be facilitated through the use of ultrasound technology. Successful injections are more common after successful aspiration of fluid is obtained. A supralateral approach into the suprapatellar pouch has been suggested to provide the most reliable access to knee joint space. In addition to administering recombinant lubricin by intra-articular injection, nucleic acids encoding recombinant lubricin (e.g., in gene therapy applications) may be administered to a synovial cavity by intra-articular injection.

[0091] For prevention of surgical adhesions, recombinant lubricins described herein are administered in the form of gel, foam, fiber or fabric. A recombinant lubricin formulated in such a manner is placed over and between damaged or exposed tissue interfaces in order to prevent adhesion formation between apposing surfaces. To be effective, the gel or film must remain in place and prevent tissue contact for a long enough time so that when the gel finally disperses and the tissues do come into contact, they will no longer have a tendency to adhere. Recombinant lubricin formulated for inhibition or prevention of adhesion formation (e.g., in the form of a membrane, fabric, foam, or gel) are evaluated for prevention of post-surgical adhesions in a rat cecal abrasion model (Goldberg et al., 1993). Compositions are placed around surgically abraded rat ceca, and compared to non-treated controls (animals whose ceca were abraded but did not receive any treatment). A reduction in the amount of adhesion formation in the rat model in the presence of recombinant lubricin formulation compared to the amount in the absence of the formulation indicates that the formulation is clinically effective to reduce tissue adhesion formation. In contexts where tissue adhesion is desired (e.g., where healing of cartilage fissures is desired), however, use of recombinant lubricin may be best avoided. Providing lubrication to cartilage surfaces impairs cartilage-cartilage integration (Schaefer et al., 2004).

[0092] Recombinant lubricins are also used to coat artificial limbs and joints prior to implantation into a mammal. For example, such devices may be dipped or bathed in a solution of a recombinant lubricin, e.g., following methods described in U.S. Pat. No. 5,709,020 or U.S. Pat. No. 5,702,456. Care should be exercised, however, in the in vivo use of recombinant lubricin in providing lubrication near a prostheses. A marked upregulation in PRG4 gene expression (i.e., MSF gene expression) has been reported to be associated with prosthesis loosening; lubricin could disturb the tight interaction between bone and prosthesis and thereby contribute to prosthesis loosening (Morawietz et al., 2003).

EXAMPLE 8

OA Model

[0093] In order to assess the efficacy of intra-articular administration of lubricin preparations, a murine model of osteoarthritis/cartilage erosion is prepared. For surgical induction of osteoarthritis, mice are anesthetized with 250 mg/kg intraperitoneal tribromoethanol (SIGMA.RTM. Chemical), and knees are prepared for aseptic surgery. A longitudinal incision medial to the patellar ligament is made, the joint capsule is opened, and the meniscotibial ligament (anchoring the medial meniscus to the tibial plateau) is identified. In a subset of animals, no further manipulation is performed, and this group is considered sham operated. In the experimental group the medial meniscotibial ligament is transected resulting in destabilization of the medial meniscus (DMM). In both sham and DMM animals, the joint capsule and subcutaneous layer are sutured closed separately and the skin is closed by application of NEXABAND.RTM. S/C tissue adhesive (Abbott, North Chicago, Ill.). Buprenorphine (BUPRENEX.RTM.; Reckitt & Coleman, Kingston-upon-Hull, UK) is administered pre- and post-operatively,

[0094] Recombinant lubricin preparations are administered by intra-articular injection using a 30 gauge needle. Injections of 5-10 microliters per knee joint are administered one week post surgery. Additional injections are optionally administered on a weekly basis. Animals are sacrificed by carbon dioxide at 4 weeks post-operatively and at 8 weeks post-operatively.

[0095] In order to assess the progression and severity of osteoarthritis, intact knee joints are placed into 4% paraformaldehyde for 24 hours, then decalcified in EDTA/polyvinylpyrrolidone for five days. Joints are embedded in paraffin and 6-.mu.m frontal sections obtained through the entire joint. Slides are stained with Safranin O-fast green and graded at 70-.mu.m intervals through the joint using a modification of a semi-quantitative scoring system (Chambers et al., 2001) in which "0"=normal cartilage; "0.5"=loss of Safranin O without structural changes; "1"=roughened articular surface and small fibrillations; "2"=fibrillation down to the layer immediately below the superficial layer and some loss of surface lamina; "3"=mild (<20%); "5"=moderate (20-80%); and "6"=severe (>80%) loss of non-calcified cartilage. Scores of "4" (erosion to bone) are not a feature of this model. All quadrants of the joint (medial tibial plateau, medial femoral condyle, lateral tibial plateau, and lateral femoral condyle) are scored separately. A minimum of 12 levels are scored by blinded observers for each knee joint. Scores are expressed as the maximum histologic score found in each joint or the summed histologic scores. The summed score represents the additive scores for each quadrant of the joint on each histologic section through the joint. This method of analysis enables assessment of severity of lesions as well as the surface area of cartilage affected with OA-like lesions (Glasson et al., 2004).

REFERENCES

[0096] (1) Chambers et al., 2001, Arthritis Rheum. 44: 1455-65; (2) Deutscher, 1990, Methods in Enzymology, Vol. 182: Guide to Protein Purification, Academic Press; (3) Espallargues and Pons, 2003, Int'l J. Tech. Assess, Health Care 19: 41-56; (4) Flannery et al., 1999, Biochem. Biophys. Res. Comm. 254: 535-41; (5) Glasson et al., 2004, Arthritis Rheum. 50: 2547-58; (6) Goldberg et al., 1993, In: Gynecologic Surgery and Adhesion Prevention, Willey-Liss, pp. 191-204; (7) Hills, 2002, J. Rheumatology 29: 200-01; (8) Ikegawa et al., 2000, Cytogenet. Cell Genet. 90: 291-297; (9) Jay et al., 2001, J. Orthopaedic Research 19: 677-87; (10) Jay et al., 2002, Glycoconjugate Journal 18: 807-15; (11) Krstenansky et al., 1987, FEBS Lett. 211: 10-16; (12) Marcelino et al., 1999, Nature Genetics 23: 319-322; (13) Merberg et al., 1993, Biology of Vitronectins and their Receptors, Pressner et al. (eds.): Elsevier Science Publishers, pp. 45-53; (14) Merrifield, 1963, J. Amer. Chem. Soc. 85: 2149-54; (15) Morawietz et al., 2003, Virchows Arch. 443: 57-66; (16) Rees et al., 2002, Matrix Biology 21: 593-602; (17) Schneerson et al., 1980, J. Exp. Med. 152: 361-76; (18) Scopes, 1994, Protein Purification: Principles and Practice (3.sup.rd edition), Springer Verlag; (19) Schaefer et al., 2004, Biorheology 41: 503-508; (20) Schumacher, 2003, Arthritis & Rheumatism 49: 413-20; (21) Tatusova and Madden, 1999, FEMS Microbiol Lett. 174: 247-50; (22) Wobig et al., 1998, Clin. Ther. 20: 410-23; and (23) Wobig et al., 1999, Clin. Ther. 21: 1549-62.

Sequence CWU 1

1

291155DNAArtificial SequenceNucleotide sequence of synthetic cDNA cassette-1. 1cgcgcccaca actccaaaag agcccgcacc taccacgaca aagtcagctc ctactacgcc 60caaagagcca gcgccgacga ctactaaaga accggcaccc accacgccta aggagccagc 120tcctactaca acgaaaccgg caccaaccac tccgg 155251PRTArtificial SequenceSynthetically generated peptide 2Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala 1 5 10 15 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala 20 25 30 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Pro Ala Pro 35 40 45 Thr Thr Pro 50 3125DNAArtificial SequenceNucleotide sequence of synthetic cDNA cassette-2. 3taaagaacca gcccctacta cgacaaagga gcctgcaccc acaaccacga agagcgcacc 60cacaacacca aaggagccgg cccctacgac tcctaaggaa cccaaaccgg caccaaccac 120tccgg 125441PRTArtificial SequenceSynthetically generated peptide 4Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala Pro Thr Thr Thr 1 5 10 15 Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Pro Lys 20 25 30 Glu Pro Lys Pro Ala Pro Thr Thr Pro 35 40 58049DNAArtificial SequencepTmed2 vector containing recombinant PRG4- Lub1 cDNA construct. 5catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgtact 60gagtcattag ggactttcca atgggttttg cccagtacat aaggtcaata ggggtgaatc 120aacaggaaag tcccattgga gccaagtaca ctgagtcaat agggactttc cattgggttt 180tgcccagtac aaaaggtcaa tagggggtga gtcaatgggt ttttcccatt attggcacgt 240acataaggtc aataggggtg agtcattggg tttttccagc caatttaatt aaaacgccat 300gtactttccc accattgacg tcaatgggct attgaaacta atgcaacgtg acctttaaac 360ggtactttcc catagctgat taatgggaaa gtaccgttct cgagccaata cacgtcaatg 420ggaagtgaaa gggcagccaa aacgtaacac cgccccggtt ttcccctgga aattccatat 480tggcacgcat tctattggct gagctgcgtt ctacgtgggt ataagaggcg cgaccagcgt 540cggtaccgtc gcagtcttcg gtctgaccac cgtagaacgc agagctcctc gctgcagccc 600aagctctgtt gggctcgcgg ttgaggacaa actcttcgcg gtctttccag tactcttgga 660tcggaaaccc gtcggcctcc gaacggtact ccgccaccga gggacctgag cgagtccgca 720tcgaccggat cggaaaacct ctcgactgtt ggggtgagta ctccctctca aaagcgggca 780tgacttctgc gctaagattg tcagtttcca aaaacgagga ggatttgata ttcacctggc 840ccgcggtgat gcctttgagg gtggccgcgt ccatctggtc agaaaagaca atctttttgt 900tgtcaagctt gaggtgtggc aggcttgaga tctggccata cacttgagtg acaatgacat 960ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcaga cttcgaattc 1020tactgagtcg acccaccatg gcatggaaaa cacttcccat ttacctgttg ttgctgctgt 1080ctgttttcgt gattcagcaa gtttcatctc aagatttatc aagctgtgca gggagatgtg 1140gggaagggta ttctagagat gccacctgca actgtgatta taactgtcaa cactacatgg 1200agtgctgccc tgatttcaag agagtctgca ctgcggagct ttcctgtaaa ggccgctgct 1260ttgagtcctt cgagagaggg agggagtgtg actgcgacgc ccaatgtaag aagtatgaca 1320agtgctgtcc cgattatgag agtttctgtg cagaagtgca taatcccaca tcaccaccat 1380cttcaaagaa agcacctcca ccttcaggag catctcaaac catcaaatca acaaccaaac 1440gttcacccaa accaccaaac aagaagaaga ctaagaaagt tatagaatca gaggaaataa 1500cagaagaaca ttctgtttct gaaaatcaag agtcctcctc cagtagcagt tcaagtagtt 1560cgtcgtcgac aatttggaaa atcaagtctt ccaaaaattc agctgctaat agagaattac 1620agaagaaact caaagtaaaa gataacaaga agaacagaac taaaaagaaa cctaccccca 1680aaccaccagt tgtagatgaa gctggaagtg gattggacaa tggtgacttc aaggtcacaa 1740ctcctgacac gtctaccacc caacacaata aagtcagcac atctcccaag atcacaacag 1800caaaaccaat aaatcccaga cccagtcttc cacctaattc tgatacatct aaagagacgt 1860ctttgacagt gaataaagag acaacagttg aaactaaaga aactactaca acaaataaac 1920agacttcaac tgatggaaaa gagaagacta cttccgctaa agagacacaa agtatagaga 1980aaacatctgc taaagattta gcacccacat ctaaagtgct ggctaaacct acacccaaag 2040ctgaaactac aaccaaaggc cctgctctca ccactcccaa ggagcccacg cccaccactc 2100ccaaggagcc tgcatctacc acacccaaag agcccacacc taccaccatc aagagcgcgc 2160ccacaactcc aaaagagccc gcacctacca cgacaaagtc agctcctact acgcccaaag 2220agccagcgcc gacgactact aaagaaccgg cacccaccac gcctaaggag ccagctccta 2280ctacaacgaa accggcacca accactccgg aaacacctcc tccaaccact tcagaggtct 2340ctactccaac taccaccaag gagcctacca ctatccacaa aagccctgat gaatcaactc 2400ctgagctttc tgcagaaccc acaccaaaag ctcttgaaaa cagtcccaag gaacctggtg 2460tacctacaac taagacgccg gcggcgacta aacctgaaat gactacaaca gctaaagaca 2520agacaacaga aagagactta cgtactacac ctgaaactac aactgctgca cctaagatga 2580caaaagagac agcaactaca acagaaaaaa ctaccgaatc caaaataaca gctacaacca 2640cacaagtaac atctaccaca actcaagata ccacaccatt caaaattact actcttaaaa 2700caactactct tgcacccaaa gtaactacaa caaaaaagac aattactacc actgagatta 2760tgaacaaacc tgaagaaaca gctaaaccaa aagacagagc tactaattct aaagcgacaa 2820ctcctaaacc tcaaaagcca accaaagcac ccaaaaaacc cacttctacc aaaaagccaa 2880aaacaatgcc tagagtgaga aaaccaaaga cgacaccaac tccccgcaag atgacatcaa 2940caatgccaga attgaaccct acctcaagaa tagcagaagc catgctccaa accaccacca 3000gacctaacca aactccaaac tccaaactag ttgaagtaaa tccaaagagt gaagatgcag 3060gtggtgctga aggagaaaca cctcatatgc ttctcaggcc ccatgtgttc atgcctgaag 3120ttactcccga catggattac ttaccgagag tacccaatca aggcattatc atcaatccca 3180tgctttccga tgagaccaat atatgcaatg gtaagccagt agatggactg actactttgc 3240gcaatgggac attagttgca ttccgaggtc attatttctg gatgctaagt ccattcagtc 3300caccatctcc agctcgcaga attactgaag tttggggtat tccttccccc attgatactg 3360tttttactag gtgcaactgt gaaggaaaaa ctttcttctt taaggattct cagtactggc 3420gttttaccaa tgatataaaa gatgcagggt accccaaacc aattttcaaa ggatttggag 3480gactaactgg acaaatagtg gcagcgcttt caacagctaa atataagaac tggcctgaat 3540ctgtgtattt tttcaagaga ggtggcagca ttcagcagta tatttataaa caggaacctg 3600tacagaagtg ccctggaaga aggcctgctc taaattatcc agtgtatgga gaaatgacac 3660aggttaggag acgtcgcttt gaacgtgcta taggaccttc tcaaacacac accatcagaa 3720ttcaatattc acctgccaga ctggcttatc aagacaaagg tgtccttcat aatgaagtta 3780aagtgagtat actgtggaga ggacttccaa atgtggttac ctcagctata tcactgccca 3840acatcagaaa acctgacggc tatgattact atgccttttc taaagatcaa tactataaca 3900ttgatgtgcc tagtagaaca gcaagagcaa ttactactcg ttctgggcag accttatcca 3960aagtctggta caactgtcct taagcggccg ccgcaaattc taacgttact ggccgaagcc 4020gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt 4080ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc 4140tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc 4200tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc 4260cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg 4320cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct 4380cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat 4440ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaacgtct 4500aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgattgc tcgagccatc 4560atggttcgac cattgaactg catcgtcgcc gtgtcccaaa atatggggat tggcaagaac 4620ggagacctac cctggcctcc gctcaggaac gagttcaagt acttccaaag aatgaccaca 4680acctcttcag tggaaggtaa acagaatctg gtgattatgg gtaggaaaac ctggttctcc 4740attcctgaga agaatcgacc tttaaaggac agaattaata tagttctcag tagagaactc 4800aaagaaccac cacgaggagc tcattttctt gccaaaagtt tggatgatgc cttaagactt 4860attgaacaac cggaattggc aagtaaagta gacatggttt ggatagtcgg aggcagttct 4920gtttaccagg aagccatgaa tcaaccaggc cacctcagac tctttgtgac aaggatcatg 4980caggaatttg aaagtgacac gtttttccca gaaattgatt tggggaaata taaacttctc 5040ccagaatacc caggcgtcct ctctgaggtc caggaggaaa aaggcatcaa gtataagttt 5100gaagtctacg agaagaaaga ctaacaggaa gatgctttca agttctctgc tcccctccta 5160aagctatgca ttttttataa gaccatggga cttttgctgg ctttagatca taatcagcca 5220taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct 5280gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta 5340caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 5400ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccccg gccaacggtc 5460tggtgacccg gctgcgagag ctcggtgtac ctgagacgcg agtaagccct tgagtcaaag 5520acgtagtcgt tgcaagtccg caccaggtac tgatcatcga tgctagaccg tgcaaaagga 5580gagcctgtaa gcgggcactc ttccgtggtc tggtggataa attcgcaagg gtatcatggc 5640ggacgaccgg ggttcgaacc ccggatccgg ccgtccgccg tgatccatcc ggttaccgcc 5700cgcgtgtcga acccaggtgt gcgacgtcag acaacggggg agcgctcctt ttggcttcct 5760tccaggcgcg gcggctgctg cgctagcttt tttggcgagc tcgaattaat tctgcattaa 5820tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5880ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5940gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 6000ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 6060cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6120ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6180accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6240caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6300gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6360tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6420agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6480actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6540gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6600aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6660gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6720aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6780atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6840gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6900atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6960ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 7020cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 7080agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 7140cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 7200tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7260agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7320gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7380gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 7440ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7500tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 7560tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7620gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7680caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7740atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 7800gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 7860tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 7920acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 7980gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg 8040agagtgcac 804962946DNAArtificial SequenceRecombinant PRG4-Lub1 cDNA construct. 6atggcatgga aaacacttcc catttacctg ttgttgctgc tgtctgtttt cgtgattcag 60caagtttcat ctcaagattt atcaagctgt gcagggagat gtggggaagg gtattctaga 120gatgccacct gcaactgtga ttataactgt caacactaca tggagtgctg ccctgatttc 180aagagagtct gcactgcgga gctttcctgt aaaggccgct gctttgagtc cttcgagaga 240gggagggagt gtgactgcga cgcccaatgt aagaagtatg acaagtgctg tcccgattat 300gagagtttct gtgcagaagt gcataatccc acatcaccac catcttcaaa gaaagcacct 360ccaccttcag gagcatctca aaccatcaaa tcaacaacca aacgttcacc caaaccacca 420aacaagaaga agactaagaa agttatagaa tcagaggaaa taacagaaga acattctgtt 480tctgaaaatc aagagtcctc ctccagtagc agttcaagta gttcgtcgtc gacaatttgg 540aaaatcaagt cttccaaaaa ttcagctgct aatagagaat tacagaagaa actcaaagta 600aaagataaca agaagaacag aactaaaaag aaacctaccc ccaaaccacc agttgtagat 660gaagctggaa gtggattgga caatggtgac ttcaaggtca caactcctga cacgtctacc 720acccaacaca ataaagtcag cacatctccc aagatcacaa cagcaaaacc aataaatccc 780agacccagtc ttccacctaa ttctgataca tctaaagaga cgtctttgac agtgaataaa 840gagacaacag ttgaaactaa agaaactact acaacaaata aacagacttc aactgatgga 900aaagagaaga ctacttccgc taaagagaca caaagtatag agaaaacatc tgctaaagat 960ttagcaccca catctaaagt gctggctaaa cctacaccca aagctgaaac tacaaccaaa 1020ggccctgctc tcaccactcc caaggagccc acgcccacca ctcccaagga gcctgcatct 1080accacaccca aagagcccac acctaccacc atcaagagcg cgcccacaac tccaaaagag 1140cccgcaccta ccacgacaaa gtcagctcct actacgccca aagagccagc gccgacgact 1200actaaagaac cggcacccac cacgcctaag gagccagctc ctactacaac gaaaccggca 1260ccaaccactc cggaaacacc tcctccaacc acttcagagg tctctactcc aactaccacc 1320aaggagccta ccactatcca caaaagccct gatgaatcaa ctcctgagct ttctgcagaa 1380cccacaccaa aagctcttga aaacagtccc aaggaacctg gtgtacctac aactaagacg 1440ccggcggcga ctaaacctga aatgactaca acagctaaag acaagacaac agaaagagac 1500ttacgtacta cacctgaaac tacaactgct gcacctaaga tgacaaaaga gacagcaact 1560acaacagaaa aaactaccga atccaaaata acagctacaa ccacacaagt aacatctacc 1620acaactcaag ataccacacc attcaaaatt actactctta aaacaactac tcttgcaccc 1680aaagtaacta caacaaaaaa gacaattact accactgaga ttatgaacaa acctgaagaa 1740acagctaaac caaaagacag agctactaat tctaaagcga caactcctaa acctcaaaag 1800ccaaccaaag cacccaaaaa acccacttct accaaaaagc caaaaacaat gcctagagtg 1860agaaaaccaa agacgacacc aactccccgc aagatgacat caacaatgcc agaattgaac 1920cctacctcaa gaatagcaga agccatgctc caaaccacca ccagacctaa ccaaactcca 1980aactccaaac tagttgaagt aaatccaaag agtgaagatg caggtggtgc tgaaggagaa 2040acacctcata tgcttctcag gccccatgtg ttcatgcctg aagttactcc cgacatggat 2100tacttaccga gagtacccaa tcaaggcatt atcatcaatc ccatgctttc cgatgagacc 2160aatatatgca atggtaagcc agtagatgga ctgactactt tgcgcaatgg gacattagtt 2220gcattccgag gtcattattt ctggatgcta agtccattca gtccaccatc tccagctcgc 2280agaattactg aagtttgggg tattccttcc cccattgata ctgtttttac taggtgcaac 2340tgtgaaggaa aaactttctt ctttaaggat tctcagtact ggcgttttac caatgatata 2400aaagatgcag ggtaccccaa accaattttc aaaggatttg gaggactaac tggacaaata 2460gtggcagcgc tttcaacagc taaatataag aactggcctg aatctgtgta ttttttcaag 2520agaggtggca gcattcagca gtatatttat aaacaggaac ctgtacagaa gtgccctgga 2580agaaggcctg ctctaaatta tccagtgtat ggagaaatga cacaggttag gagacgtcgc 2640tttgaacgtg ctataggacc ttctcaaaca cacaccatca gaattcaata ttcacctgcc 2700agactggctt atcaagacaa aggtgtcctt cataatgaag ttaaagtgag tatactgtgg 2760agaggacttc caaatgtggt tacctcagct atatcactgc ccaacatcag aaaacctgac 2820ggctatgatt actatgcctt ttctaaagat caatactata acattgatgt gcctagtaga 2880acagcaagag caattactac tcgttctggg cagaccttat ccaaagtctg gtacaactgt 2940ccttaa 29467981PRTArtificial SequenceAmino acid sequence of entire PRG4-LUB1 protein. 7Met Ala Trp Lys Thr Leu Pro Ile Tyr Leu Leu Leu Leu Leu Ser Val 1 5 10 15 Phe Val Ile Gln Gln Val Ser Ser Gln Asp Leu Ser Ser Cys Ala Gly 20 25 30 Arg Cys Gly Glu Gly Tyr Ser Arg Asp Ala Thr Cys Asn Cys Asp Tyr 35 40 45 Asn Cys Gln His Tyr Met Glu Cys Cys Pro Asp Phe Lys Arg Val Cys 50 55 60 Thr Ala Glu Leu Ser Cys Lys Gly Arg Cys Phe Glu Ser Phe Glu Arg 65 70 75 80 Gly Arg Glu Cys Asp Cys Asp Ala Gln Cys Lys Lys Tyr Asp Lys Cys 85 90 95 Cys Pro Asp Tyr Glu Ser Phe Cys Ala Glu Val His Asn Pro Thr Ser 100 105 110 Pro Pro Ser Ser Lys Lys Ala Pro Pro Pro Ser Gly Ala Ser Gln Thr 115 120 125 Ile Lys Ser Thr Thr Lys Arg Ser Pro Lys Pro Pro Asn Lys Lys Lys 130 135 140 Thr Lys Lys Val Ile Glu Ser Glu Glu Ile Thr Glu Glu His Ser Val 145 150 155 160 Ser Glu Asn Gln Glu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 165 170 175 Ser Thr Ile Trp Lys Ile Lys Ser Ser Lys Asn Ser Ala Ala Asn Arg 180 185 190 Glu Leu Gln Lys Lys Leu Lys Val Lys Asp Asn Lys Lys Asn Arg Thr 195 200 205 Lys Lys Lys Pro Thr Pro Lys Pro Pro Val Val Asp Glu Ala Gly Ser 210 215 220 Gly Leu Asp Asn Gly Asp Phe Lys Val Thr Thr Pro Asp Thr Ser Thr 225 230 235 240 Thr Gln His Asn Lys Val Ser Thr Ser Pro Lys Ile Thr Thr Ala Lys 245 250 255 Pro Ile Asn Pro Arg Pro Ser Leu Pro Pro Asn Ser Asp Thr Ser Lys 260 265 270 Glu Thr Ser Leu Thr Val Asn Lys Glu Thr Thr Val Glu Thr Lys Glu 275 280 285 Thr Thr Thr Thr Asn Lys Gln Thr Ser Thr Asp Gly Lys Glu Lys Thr 290 295 300 Thr Ser Ala Lys Glu Thr Gln Ser Ile Glu Lys Thr Ser Ala Lys Asp 305 310 315 320 Leu Ala Pro Thr Ser Lys Val Leu Ala Lys Pro Thr Pro Lys Ala Glu 325 330 335 Thr Thr Thr Lys Gly Pro Ala Leu Thr Thr Pro Lys Glu Pro Thr Pro 340 345 350 Thr Thr Pro Lys Glu Pro Ala Ser Thr Thr Pro Lys Glu Pro Thr Pro 355 360 365 Thr Thr Ile Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr 370 375 380 Thr Thr Lys Ser Ala

Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 385 390 395 400 Thr Lys Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 405 410 415 Thr Lys Pro Ala Pro Thr Thr Pro Glu Thr Pro Pro Pro Thr Thr Ser 420 425 430 Glu Val Ser Thr Pro Thr Thr Thr Lys Glu Pro Thr Thr Ile His Lys 435 440 445 Ser Pro Asp Glu Ser Thr Pro Glu Leu Ser Ala Glu Pro Thr Pro Lys 450 455 460 Ala Leu Glu Asn Ser Pro Lys Glu Pro Gly Val Pro Thr Thr Lys Thr 465 470 475 480 Pro Ala Ala Thr Lys Pro Glu Met Thr Thr Thr Ala Lys Asp Lys Thr 485 490 495 Thr Glu Arg Asp Leu Arg Thr Thr Pro Glu Thr Thr Thr Ala Ala Pro 500 505 510 Lys Met Thr Lys Glu Thr Ala Thr Thr Thr Glu Lys Thr Thr Glu Ser 515 520 525 Lys Ile Thr Ala Thr Thr Thr Gln Val Thr Ser Thr Thr Thr Gln Asp 530 535 540 Thr Thr Pro Phe Lys Ile Thr Thr Leu Lys Thr Thr Thr Leu Ala Pro 545 550 555 560 Lys Val Thr Thr Thr Lys Lys Thr Ile Thr Thr Thr Glu Ile Met Asn 565 570 575 Lys Pro Glu Glu Thr Ala Lys Pro Lys Asp Arg Ala Thr Asn Ser Lys 580 585 590 Ala Thr Thr Pro Lys Pro Gln Lys Pro Thr Lys Ala Pro Lys Lys Pro 595 600 605 Thr Ser Thr Lys Lys Pro Lys Thr Met Pro Arg Val Arg Lys Pro Lys 610 615 620 Thr Thr Pro Thr Pro Arg Lys Met Thr Ser Thr Met Pro Glu Leu Asn 625 630 635 640 Pro Thr Ser Arg Ile Ala Glu Ala Met Leu Gln Thr Thr Thr Arg Pro 645 650 655 Asn Gln Thr Pro Asn Ser Lys Leu Val Glu Val Asn Pro Lys Ser Glu 660 665 670 Asp Ala Gly Gly Ala Glu Gly Glu Thr Pro His Met Leu Leu Arg Pro 675 680 685 His Val Phe Met Pro Glu Val Thr Pro Asp Met Asp Tyr Leu Pro Arg 690 695 700 Val Pro Asn Gln Gly Ile Ile Ile Asn Pro Met Leu Ser Asp Glu Thr 705 710 715 720 Asn Ile Cys Asn Gly Lys Pro Val Asp Gly Leu Thr Thr Leu Arg Asn 725 730 735 Gly Thr Leu Val Ala Phe Arg Gly His Tyr Phe Trp Met Leu Ser Pro 740 745 750 Phe Ser Pro Pro Ser Pro Ala Arg Arg Ile Thr Glu Val Trp Gly Ile 755 760 765 Pro Ser Pro Ile Asp Thr Val Phe Thr Arg Cys Asn Cys Glu Gly Lys 770 775 780 Thr Phe Phe Phe Lys Asp Ser Gln Tyr Trp Arg Phe Thr Asn Asp Ile 785 790 795 800 Lys Asp Ala Gly Tyr Pro Lys Pro Ile Phe Lys Gly Phe Gly Gly Leu 805 810 815 Thr Gly Gln Ile Val Ala Ala Leu Ser Thr Ala Lys Tyr Lys Asn Trp 820 825 830 Pro Glu Ser Val Tyr Phe Phe Lys Arg Gly Gly Ser Ile Gln Gln Tyr 835 840 845 Ile Tyr Lys Gln Glu Pro Val Gln Lys Cys Pro Gly Arg Arg Pro Ala 850 855 860 Leu Asn Tyr Pro Val Tyr Gly Glu Met Thr Gln Val Arg Arg Arg Arg 865 870 875 880 Phe Glu Arg Ala Ile Gly Pro Ser Gln Thr His Thr Ile Arg Ile Gln 885 890 895 Tyr Ser Pro Ala Arg Leu Ala Tyr Gln Asp Lys Gly Val Leu His Asn 900 905 910 Glu Val Lys Val Ser Ile Leu Trp Arg Gly Leu Pro Asn Val Val Thr 915 920 925 Ser Ala Ile Ser Leu Pro Asn Ile Arg Lys Pro Asp Gly Tyr Asp Tyr 930 935 940 Tyr Ala Phe Ser Lys Asp Gln Tyr Tyr Asn Ile Asp Val Pro Ser Arg 945 950 955 960 Thr Ala Arg Ala Ile Thr Thr Arg Ser Gly Gln Thr Leu Ser Lys Val 965 970 975 Trp Tyr Asn Cys Pro 980 8157DNAArtificial SequenceLub1 DNA insert from synthetic cDNA cassette-1. 8gcgcgcccac aactccaaaa gagcccgcac ctaccacgac aaagtcagct cctactacgc 60ccaaagagcc agcgccgacg actactaaag aaccggcacc caccacgcct aaggagccag 120ctcctactac aacgaaaccg gcaccaacca ctccgga 157951PRTArtificial Sequence51 amino acids encoded by Lub1 DNA insert (4 KEPAPTT sequences between S373 to E425 in SEQ ID NO 7). 9Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala 1 5 10 15 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala 20 25 30 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Pro Ala Pro 35 40 45 Thr Thr Pro 50 103024DNAArtificial SequenceRecombinant PRG4-Lub2 cDNA construct. 10atggcatgga aaacacttcc catttacctg ttgttgctgc tgtctgtttt cgtgattcag 60caagtttcat ctcaagattt atcaagctgt gcagggagat gtggggaagg gtattctaga 120gatgccacct gcaactgtga ttataactgt caacactaca tggagtgctg ccctgatttc 180aagagagtct gcactgcgga gctttcctgt aaaggccgct gctttgagtc cttcgagaga 240gggagggagt gtgactgcga cgcccaatgt aagaagtatg acaagtgctg tcccgattat 300gagagtttct gtgcagaagt gcataatccc acatcaccac catcttcaaa gaaagcacct 360ccaccttcag gagcatctca aaccatcaaa tcaacaacca aacgttcacc caaaccacca 420aacaagaaga agactaagaa agttatagaa tcagaggaaa taacagaaga acattctgtt 480tctgaaaatc aagagtcctc ctccagtagc agttcaagta gttcgtcgtc gacaatttgg 540aaaatcaagt cttccaaaaa ttcagctgct aatagagaat tacagaagaa actcaaagta 600aaagataaca agaagaacag aactaaaaag aaacctaccc ccaaaccacc agttgtagat 660gaagctggaa gtggattgga caatggtgac ttcaaggtca caactcctga cacgtctacc 720acccaacaca ataaagtcag cacatctccc aagatcacaa cagcaaaacc aataaatccc 780agacccagtc ttccacctaa ttctgataca tctaaagaga cgtctttgac agtgaataaa 840gagacaacag ttgaaactaa agaaactact acaacaaata aacagacttc aactgatgga 900aaagagaaga ctacttccgc taaagagaca caaagtatag agaaaacatc tgctaaagat 960ttagcaccca catctaaagt gctggctaaa cctacaccca aagctgaaac tacaaccaaa 1020ggccctgctc tcaccactcc caaggagccc acgcccacca ctcccaagga gcctgcatct 1080accacaccca aagagcccac acctaccacc atcaagagcg cgcccacaac tccaaaagag 1140cccgcaccta ccacgacaaa gtcagctcct actacgccca aagagccagc gccgacgact 1200actaaagaac cggcacccac cacgcctaaa gaaccagccc ctactacgac aaaggagcct 1260gcacccacaa ccacgaagag cgcacccaca acaccaaagg agccggcccc tacgactcct 1320aaggaaccca aaccggcacc aaccactccg gaaacacctc ctccaaccac ttcagaggtc 1380tctactccaa ctaccaccaa ggagcctacc actatccaca aaagccctga tgaatcaact 1440cctgagcttt ctgcagaacc cacaccaaaa gctcttgaaa acagtcccaa ggaacctggt 1500gtacctacaa ctaagacgcc ggcggcgact aaacctgaaa tgactacaac agctaaagac 1560aagacaacag aaagagactt acgtactaca cctgaaacta caactgctgc acctaagatg 1620acaaaagaga cagcaactac aacagaaaaa actaccgaat ccaaaataac agctacaacc 1680acacaagtaa catctaccac aactcaagat accacaccat tcaaaattac tactcttaaa 1740acaactactc ttgcacccaa agtaactaca acaaaaaaga caattactac cactgagatt 1800atgaacaaac ctgaagaaac agctaaacca aaagacagag ctactaattc taaagcgaca 1860actcctaaac ctcaaaagcc aaccaaagca cccaaaaaac ccacttctac caaaaagcca 1920aaaacaatgc ctagagtgag aaaaccaaag acgacaccaa ctccccgcaa gatgacatca 1980acaatgccag aattgaaccc tacctcaaga atagcagaag ccatgctcca aaccaccacc 2040agacctaacc aaactccaaa ctccaaacta gttgaagtaa atccaaagag tgaagatgca 2100ggtggtgctg aaggagaaac acctcatatg cttctcaggc cccatgtgtt catgcctgaa 2160gttactcccg acatggatta cttaccgaga gtacccaatc aaggcattat catcaatccc 2220atgctttccg atgagaccaa tatatgcaat ggtaagccag tagatggact gactactttg 2280cgcaatggga cattagttgc attccgaggt cattatttct ggatgctaag tccattcagt 2340ccaccatctc cagctcgcag aattactgaa gtttggggta ttccttcccc cattgatact 2400gtttttacta ggtgcaactg tgaaggaaaa actttcttct ttaaggattc tcagtactgg 2460cgttttacca atgatataaa agatgcaggg taccccaaac caattttcaa aggatttgga 2520ggactaactg gacaaatagt ggcagcgctt tcaacagcta aatataagaa ctggcctgaa 2580tctgtgtatt ttttcaagag aggtggcagc attcagcagt atatttataa acaggaacct 2640gtacagaagt gccctggaag aaggcctgct ctaaattatc cagtgtatgg agaaatgaca 2700caggttagga gacgtcgctt tgaacgtgct ataggacctt ctcaaacaca caccatcaga 2760attcaatatt cacctgccag actggcttat caagacaaag gtgtccttca taatgaagtt 2820aaagtgagta tactgtggag aggacttcca aatgtggtta cctcagctat atcactgccc 2880aacatcagaa aacctgacgg ctatgattac tatgcctttt ctaaagatca atactataac 2940attgatgtgc ctagtagaac agcaagagca attactactc gttctgggca gaccttatcc 3000aaagtctggt acaactgtcc ttaa 3024111007PRTArtificial SequenceAmino acid sequence of entire PRG4-LUB2 protein. 11Met Ala Trp Lys Thr Leu Pro Ile Tyr Leu Leu Leu Leu Leu Ser Val 1 5 10 15 Phe Val Ile Gln Gln Val Ser Ser Gln Asp Leu Ser Ser Cys Ala Gly 20 25 30 Arg Cys Gly Glu Gly Tyr Ser Arg Asp Ala Thr Cys Asn Cys Asp Tyr 35 40 45 Asn Cys Gln His Tyr Met Glu Cys Cys Pro Asp Phe Lys Arg Val Cys 50 55 60 Thr Ala Glu Leu Ser Cys Lys Gly Arg Cys Phe Glu Ser Phe Glu Arg 65 70 75 80 Gly Arg Glu Cys Asp Cys Asp Ala Gln Cys Lys Lys Tyr Asp Lys Cys 85 90 95 Cys Pro Asp Tyr Glu Ser Phe Cys Ala Glu Val His Asn Pro Thr Ser 100 105 110 Pro Pro Ser Ser Lys Lys Ala Pro Pro Pro Ser Gly Ala Ser Gln Thr 115 120 125 Ile Lys Ser Thr Thr Lys Arg Ser Pro Lys Pro Pro Asn Lys Lys Lys 130 135 140 Thr Lys Lys Val Ile Glu Ser Glu Glu Ile Thr Glu Glu His Ser Val 145 150 155 160 Ser Glu Asn Gln Glu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 165 170 175 Ser Thr Ile Trp Lys Ile Lys Ser Ser Lys Asn Ser Ala Ala Asn Arg 180 185 190 Glu Leu Gln Lys Lys Leu Lys Val Lys Asp Asn Lys Lys Asn Arg Thr 195 200 205 Lys Lys Lys Pro Thr Pro Lys Pro Pro Val Val Asp Glu Ala Gly Ser 210 215 220 Gly Leu Asp Asn Gly Asp Phe Lys Val Thr Thr Pro Asp Thr Ser Thr 225 230 235 240 Thr Gln His Asn Lys Val Ser Thr Ser Pro Lys Ile Thr Thr Ala Lys 245 250 255 Pro Ile Asn Pro Arg Pro Ser Leu Pro Pro Asn Ser Asp Thr Ser Lys 260 265 270 Glu Thr Ser Leu Thr Val Asn Lys Glu Thr Thr Val Glu Thr Lys Glu 275 280 285 Thr Thr Thr Thr Asn Lys Gln Thr Ser Thr Asp Gly Lys Glu Lys Thr 290 295 300 Thr Ser Ala Lys Glu Thr Gln Ser Ile Glu Lys Thr Ser Ala Lys Asp 305 310 315 320 Leu Ala Pro Thr Ser Lys Val Leu Ala Lys Pro Thr Pro Lys Ala Glu 325 330 335 Thr Thr Thr Lys Gly Pro Ala Leu Thr Thr Pro Lys Glu Pro Thr Pro 340 345 350 Thr Thr Pro Lys Glu Pro Ala Ser Thr Thr Pro Lys Glu Pro Thr Pro 355 360 365 Thr Thr Ile Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr 370 375 380 Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 385 390 395 400 Thr Lys Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 405 410 415 Thr Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro 420 425 430 Lys Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Lys Pro Ala Pro Thr 435 440 445 Thr Pro Glu Thr Pro Pro Pro Thr Thr Ser Glu Val Ser Thr Pro Thr 450 455 460 Thr Thr Lys Glu Pro Thr Thr Ile His Lys Ser Pro Asp Glu Ser Thr 465 470 475 480 Pro Glu Leu Ser Ala Glu Pro Thr Pro Lys Ala Leu Glu Asn Ser Pro 485 490 495 Lys Glu Pro Gly Val Pro Thr Thr Lys Thr Pro Ala Ala Thr Lys Pro 500 505 510 Glu Met Thr Thr Thr Ala Lys Asp Lys Thr Thr Glu Arg Asp Leu Arg 515 520 525 Thr Thr Pro Glu Thr Thr Thr Ala Ala Pro Lys Met Thr Lys Glu Thr 530 535 540 Ala Thr Thr Thr Glu Lys Thr Thr Glu Ser Lys Ile Thr Ala Thr Thr 545 550 555 560 Thr Gln Val Thr Ser Thr Thr Thr Gln Asp Thr Thr Pro Phe Lys Ile 565 570 575 Thr Thr Leu Lys Thr Thr Thr Leu Ala Pro Lys Val Thr Thr Thr Lys 580 585 590 Lys Thr Ile Thr Thr Thr Glu Ile Met Asn Lys Pro Glu Glu Thr Ala 595 600 605 Lys Pro Lys Asp Arg Ala Thr Asn Ser Lys Ala Thr Thr Pro Lys Pro 610 615 620 Gln Lys Pro Thr Lys Ala Pro Lys Lys Pro Thr Ser Thr Lys Lys Pro 625 630 635 640 Lys Thr Met Pro Arg Val Arg Lys Pro Lys Thr Thr Pro Thr Pro Arg 645 650 655 Lys Met Thr Ser Thr Met Pro Glu Leu Asn Pro Thr Ser Arg Ile Ala 660 665 670 Glu Ala Met Leu Gln Thr Thr Thr Arg Pro Asn Gln Thr Pro Asn Ser 675 680 685 Lys Leu Val Glu Val Asn Pro Lys Ser Glu Asp Ala Gly Gly Ala Glu 690 695 700 Gly Glu Thr Pro His Met Leu Leu Arg Pro His Val Phe Met Pro Glu 705 710 715 720 Val Thr Pro Asp Met Asp Tyr Leu Pro Arg Val Pro Asn Gln Gly Ile 725 730 735 Ile Ile Asn Pro Met Leu Ser Asp Glu Thr Asn Ile Cys Asn Gly Lys 740 745 750 Pro Val Asp Gly Leu Thr Thr Leu Arg Asn Gly Thr Leu Val Ala Phe 755 760 765 Arg Gly His Tyr Phe Trp Met Leu Ser Pro Phe Ser Pro Pro Ser Pro 770 775 780 Ala Arg Arg Ile Thr Glu Val Trp Gly Ile Pro Ser Pro Ile Asp Thr 785 790 795 800 Val Phe Thr Arg Cys Asn Cys Glu Gly Lys Thr Phe Phe Phe Lys Asp 805 810 815 Ser Gln Tyr Trp Arg Phe Thr Asn Asp Ile Lys Asp Ala Gly Tyr Pro 820 825 830 Lys Pro Ile Phe Lys Gly Phe Gly Gly Leu Thr Gly Gln Ile Val Ala 835 840 845 Ala Leu Ser Thr Ala Lys Tyr Lys Asn Trp Pro Glu Ser Val Tyr Phe 850 855 860 Phe Lys Arg Gly Gly Ser Ile Gln Gln Tyr Ile Tyr Lys Gln Glu Pro 865 870 875 880 Val Gln Lys Cys Pro Gly Arg Arg Pro Ala Leu Asn Tyr Pro Val Tyr 885 890 895 Gly Glu Met Thr Gln Val Arg Arg Arg Arg Phe Glu Arg Ala Ile Gly 900 905 910 Pro Ser Gln Thr His Thr Ile Arg Ile Gln Tyr Ser Pro Ala Arg Leu 915 920 925 Ala Tyr Gln Asp Lys Gly Val Leu His Asn Glu Val Lys Val Ser Ile 930 935 940 Leu Trp Arg Gly Leu Pro Asn Val Val Thr Ser Ala Ile Ser Leu Pro 945 950 955 960 Asn Ile Arg Lys Pro Asp Gly Tyr Asp Tyr Tyr Ala Phe Ser Lys Asp 965 970 975 Gln Tyr Tyr Asn Ile Asp Val Pro Ser Arg Thr Ala Arg Ala Ile Thr 980 985 990 Thr Arg Ser Gly Gln Thr Leu Ser Lys Val Trp Tyr Asn Cys Pro 995 1000 1005 12235DNAArtificial SequenceLub2 DNA insert from synthetic cDNA cassette-1 and one synthetic cDNA cassette-2 sequence. 12gcgcgcccac aactccaaaa gagcccgcac ctaccacgac aaagtcagct cctactacgc 60ccaaagagcc agcgccgacg actactaaag aaccggcacc caccacgcct aaagaaccag 120cccctactac gacaaaggag cctgcaccca caaccacgaa gagcgcaccc acaacaccaa 180aggagccggc ccctacgact cctaaggaac ccaaaccggc accaaccact ccgga 2351377PRTArtificial Sequence77 amino acids encoded by Lub2 DNA insert (6 KEPAPTT sequences between S373 and E451 in SEQ ID NO 11). 13Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala 1 5 10 15 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala 20 25 30 Pro Thr Thr Pro Lys Glu Pro Ala

Pro Thr Thr Thr Lys Glu Pro Ala 35 40 45 Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro 50 55 60 Thr Thr Pro Lys Glu Pro Lys Pro Ala Pro Thr Thr Pro 65 70 75 143117DNAArtificial SequenceRecombinant PRG4-Lub3 cDNA construct. 14atggcatgga aaacacttcc catttacctg ttgttgctgc tgtctgtttt cgtgattcag 60caagtttcat ctcaagattt atcaagctgt gcagggagat gtggggaagg gtattctaga 120gatgccacct gcaactgtga ttataactgt caacactaca tggagtgctg ccctgatttc 180aagagagtct gcactgcgga gctttcctgt aaaggccgct gctttgagtc cttcgagaga 240gggagggagt gtgactgcga cgcccaatgt aagaagtatg acaagtgctg tcccgattat 300gagagtttct gtgcagaagt gcataatccc acatcaccac catcttcaaa gaaagcacct 360ccaccttcag gagcatctca aaccatcaaa tcaacaacca aacgttcacc caaaccacca 420aacaagaaga agactaagaa agttatagaa tcagaggaaa taacagaaga acattctgtt 480tctgaaaatc aagagtcctc ctccagtagc agttcaagta gttcgtcgtc gacaatttgg 540aaaatcaagt cttccaaaaa ttcagctgct aatagagaat tacagaagaa actcaaagta 600aaagataaca agaagaacag aactaaaaag aaacctaccc ccaaaccacc agttgtagat 660gaagctggaa gtggattgga caatggtgac ttcaaggtca caactcctga cacgtctacc 720acccaacaca ataaagtcag cacatctccc aagatcacaa cagcaaaacc aataaatccc 780agacccagtc ttccacctaa ttctgataca tctaaagaga cgtctttgac agtgaataaa 840gagacaacag ttgaaactaa agaaactact acaacaaata aacagacttc aactgatgga 900aaagagaaga ctacttccgc taaagagaca caaagtatag agaaaacatc tgctaaagat 960ttagcaccca catctaaagt gctggctaaa cctacaccca aagctgaaac tacaaccaaa 1020ggccctgctc tcaccactcc caaggagccc acgcccacca ctcccaagga gcctgcatct 1080accacaccca aagagcccac acctaccacc atcaagagcg cgcccacaac tccaaaagag 1140cccgcaccta ccacgacaaa gtcagctcct actacgccca aagagccagc gccgacgact 1200actaaagaac cggcacccac cacgcctaaa gaaccagccc ctactacgac aaaggagcct 1260gcacccacaa ccacgaagag cgcacccaca acaccaaagg agccggcccc tacgactcct 1320aaagaaccag cccctactac gacaaaggag cctgcaccca caaccacgaa gagcgcaccc 1380acaacaccaa aggagccggc ccctacgact cctaaggaac ccaaaccggc accaaccact 1440ccggaaacac ctcctccaac cacttcagag gtctctactc caactaccac caaggagcct 1500accactatcc acaaaagccc tgatgaatca actcctgagc tttctgcaga acccacacca 1560aaagctcttg aaaacagtcc caaggaacct ggtgtaccta caactaagac gccggcggcg 1620actaaacctg aaatgactac aacagctaaa gacaagacaa cagaaagaga cttacgtact 1680acacctgaaa ctacaactgc tgcacctaag atgacaaaag agacagcaac tacaacagaa 1740aaaactaccg aatccaaaat aacagctaca accacacaag taacatctac cacaactcaa 1800gataccacac cattcaaaat tactactctt aaaacaacta ctcttgcacc caaagtaact 1860acaacaaaaa agacaattac taccactgag attatgaaca aacctgaaga aacagctaaa 1920ccaaaagaca gagctactaa ttctaaagcg acaactccta aacctcaaaa gccaaccaaa 1980gcacccaaaa aacccacttc taccaaaaag ccaaaaacaa tgcctagagt gagaaaacca 2040aagacgacac caactccccg caagatgaca tcaacaatgc cagaattgaa ccctacctca 2100agaatagcag aagccatgct ccaaaccacc accagaccta accaaactcc aaactccaaa 2160ctagttgaag taaatccaaa gagtgaagat gcaggtggtg ctgaaggaga aacacctcat 2220atgcttctca ggccccatgt gttcatgcct gaagttactc ccgacatgga ttacttaccg 2280agagtaccca atcaaggcat tatcatcaat cccatgcttt ccgatgagac caatatatgc 2340aatggtaagc cagtagatgg actgactact ttgcgcaatg ggacattagt tgcattccga 2400ggtcattatt tctggatgct aagtccattc agtccaccat ctccagctcg cagaattact 2460gaagtttggg gtattccttc ccccattgat actgttttta ctaggtgcaa ctgtgaagga 2520aaaactttct tctttaagga ttctcagtac tggcgtttta ccaatgatat aaaagatgca 2580gggtacccca aaccaatttt caaaggattt ggaggactaa ctggacaaat agtggcagcg 2640ctttcaacag ctaaatataa gaactggcct gaatctgtgt attttttcaa gagaggtggc 2700agcattcagc agtatattta taaacaggaa cctgtacaga agtgccctgg aagaaggcct 2760gctctaaatt atccagtgta tggagaaatg acacaggtta ggagacgtcg ctttgaacgt 2820gctataggac cttctcaaac acacaccatc agaattcaat attcacctgc cagactggct 2880tatcaagaca aaggtgtcct tcataatgaa gttaaagtga gtatactgtg gagaggactt 2940ccaaatgtgg ttacctcagc tatatcactg cccaacatca gaaaacctga cggctatgat 3000tactatgcct tttctaaaga tcaatactat aacattgatg tgcctagtag aacagcaaga 3060gcaattacta ctcgttctgg gcagacctta tccaaagtct ggtacaactg tccttaa 3117151038PRTArtificial Sequenceamino acid sequence of entire PRG4-LUB3 protein 15Met Ala Trp Lys Thr Leu Pro Ile Tyr Leu Leu Leu Leu Leu Ser Val 1 5 10 15 Phe Val Ile Gln Gln Val Ser Ser Gln Asp Leu Ser Ser Cys Ala Gly 20 25 30 Arg Cys Gly Glu Gly Tyr Ser Arg Asp Ala Thr Cys Asn Cys Asp Tyr 35 40 45 Asn Cys Gln His Tyr Met Glu Cys Cys Pro Asp Phe Lys Arg Val Cys 50 55 60 Thr Ala Glu Leu Ser Cys Lys Gly Arg Cys Phe Glu Ser Phe Glu Arg 65 70 75 80 Gly Arg Glu Cys Asp Cys Asp Ala Gln Cys Lys Lys Tyr Asp Lys Cys 85 90 95 Cys Pro Asp Tyr Glu Ser Phe Cys Ala Glu Val His Asn Pro Thr Ser 100 105 110 Pro Pro Ser Ser Lys Lys Ala Pro Pro Pro Ser Gly Ala Ser Gln Thr 115 120 125 Ile Lys Ser Thr Thr Lys Arg Ser Pro Lys Pro Pro Asn Lys Lys Lys 130 135 140 Thr Lys Lys Val Ile Glu Ser Glu Glu Ile Thr Glu Glu His Ser Val 145 150 155 160 Ser Glu Asn Gln Glu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 165 170 175 Ser Thr Ile Trp Lys Ile Lys Ser Ser Lys Asn Ser Ala Ala Asn Arg 180 185 190 Glu Leu Gln Lys Lys Leu Lys Val Lys Asp Asn Lys Lys Asn Arg Thr 195 200 205 Lys Lys Lys Pro Thr Pro Lys Pro Pro Val Val Asp Glu Ala Gly Ser 210 215 220 Gly Leu Asp Asn Gly Asp Phe Lys Val Thr Thr Pro Asp Thr Ser Thr 225 230 235 240 Thr Gln His Asn Lys Val Ser Thr Ser Pro Lys Ile Thr Thr Ala Lys 245 250 255 Pro Ile Asn Pro Arg Pro Ser Leu Pro Pro Asn Ser Asp Thr Ser Lys 260 265 270 Glu Thr Ser Leu Thr Val Asn Lys Glu Thr Thr Val Glu Thr Lys Glu 275 280 285 Thr Thr Thr Thr Asn Lys Gln Thr Ser Thr Asp Gly Lys Glu Lys Thr 290 295 300 Thr Ser Ala Lys Glu Thr Gln Ser Ile Glu Lys Thr Ser Ala Lys Asp 305 310 315 320 Leu Ala Pro Thr Ser Lys Val Leu Ala Lys Pro Thr Pro Lys Ala Glu 325 330 335 Thr Thr Thr Lys Gly Pro Ala Leu Thr Thr Pro Lys Glu Pro Thr Pro 340 345 350 Thr Thr Pro Lys Glu Pro Ala Ser Thr Thr Pro Lys Glu Pro Thr Pro 355 360 365 Thr Thr Ile Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr 370 375 380 Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 385 390 395 400 Thr Lys Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 405 410 415 Thr Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro 420 425 430 Lys Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr 435 440 445 Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys 450 455 460 Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Lys Pro Ala Pro Thr Thr 465 470 475 480 Pro Glu Thr Pro Pro Pro Thr Thr Ser Glu Val Ser Thr Pro Thr Thr 485 490 495 Thr Lys Glu Pro Thr Thr Ile His Lys Ser Pro Asp Glu Ser Thr Pro 500 505 510 Glu Leu Ser Ala Glu Pro Thr Pro Lys Ala Leu Glu Asn Ser Pro Lys 515 520 525 Glu Pro Gly Val Pro Thr Thr Lys Thr Pro Ala Ala Thr Lys Pro Glu 530 535 540 Met Thr Thr Thr Ala Lys Asp Lys Thr Thr Glu Arg Asp Leu Arg Thr 545 550 555 560 Thr Pro Glu Thr Thr Thr Ala Ala Pro Lys Met Thr Lys Glu Thr Ala 565 570 575 Thr Thr Thr Glu Lys Thr Thr Glu Ser Lys Ile Thr Ala Thr Thr Thr 580 585 590 Gln Val Thr Ser Thr Thr Thr Gln Asp Thr Thr Pro Phe Lys Ile Thr 595 600 605 Thr Leu Lys Thr Thr Thr Leu Ala Pro Lys Val Thr Thr Thr Lys Lys 610 615 620 Thr Ile Thr Thr Thr Glu Ile Met Asn Lys Pro Glu Glu Thr Ala Lys 625 630 635 640 Pro Lys Asp Arg Ala Thr Asn Ser Lys Ala Thr Thr Pro Lys Pro Gln 645 650 655 Lys Pro Thr Lys Ala Pro Lys Lys Pro Thr Ser Thr Lys Lys Pro Lys 660 665 670 Thr Met Pro Arg Val Arg Lys Pro Lys Thr Thr Pro Thr Pro Arg Lys 675 680 685 Met Thr Ser Thr Met Pro Glu Leu Asn Pro Thr Ser Arg Ile Ala Glu 690 695 700 Ala Met Leu Gln Thr Thr Thr Arg Pro Asn Gln Thr Pro Asn Ser Lys 705 710 715 720 Leu Val Glu Val Asn Pro Lys Ser Glu Asp Ala Gly Gly Ala Glu Gly 725 730 735 Glu Thr Pro His Met Leu Leu Arg Pro His Val Phe Met Pro Glu Val 740 745 750 Thr Pro Asp Met Asp Tyr Leu Pro Arg Val Pro Asn Gln Gly Ile Ile 755 760 765 Ile Asn Pro Met Leu Ser Asp Glu Thr Asn Ile Cys Asn Gly Lys Pro 770 775 780 Val Asp Gly Leu Thr Thr Leu Arg Asn Gly Thr Leu Val Ala Phe Arg 785 790 795 800 Gly His Tyr Phe Trp Met Leu Ser Pro Phe Ser Pro Pro Ser Pro Ala 805 810 815 Arg Arg Ile Thr Glu Val Trp Gly Ile Pro Ser Pro Ile Asp Thr Val 820 825 830 Phe Thr Arg Cys Asn Cys Glu Gly Lys Thr Phe Phe Phe Lys Asp Ser 835 840 845 Gln Tyr Trp Arg Phe Thr Asn Asp Ile Lys Asp Ala Gly Tyr Pro Lys 850 855 860 Pro Ile Phe Lys Gly Phe Gly Gly Leu Thr Gly Gln Ile Val Ala Ala 865 870 875 880 Leu Ser Thr Ala Lys Tyr Lys Asn Trp Pro Glu Ser Val Tyr Phe Phe 885 890 895 Lys Arg Gly Gly Ser Ile Gln Gln Tyr Ile Tyr Lys Gln Glu Pro Val 900 905 910 Gln Lys Cys Pro Gly Arg Arg Pro Ala Leu Asn Tyr Pro Val Tyr Gly 915 920 925 Glu Met Thr Gln Val Arg Arg Arg Arg Phe Glu Arg Ala Ile Gly Pro 930 935 940 Ser Gln Thr His Thr Ile Arg Ile Gln Tyr Ser Pro Ala Arg Leu Ala 945 950 955 960 Tyr Gln Asp Lys Gly Val Leu His Asn Glu Val Lys Val Ser Ile Leu 965 970 975 Trp Arg Gly Leu Pro Asn Val Val Thr Ser Ala Ile Ser Leu Pro Asn 980 985 990 Ile Arg Lys Pro Asp Gly Tyr Asp Tyr Tyr Ala Phe Ser Lys Asp Gln 995 1000 1005 Tyr Tyr Asn Ile Asp Val Pro Ser Arg Thr Ala Arg Ala Ile Thr 1010 1015 1020 Thr Arg Ser Gly Gln Thr Leu Ser Lys Val Trp Tyr Asn Cys Pro 1025 1030 1035 16328DNAArtificial SequenceLub3 DNA insert from synthetic cDNA cassette-1 and two synthetic cDNA cassette-2 sequences. 16gcgcgcccac aactccaaaa gagcccgcac ctaccacgac aaagtcagct cctactacgc 60ccaaagagcc agcgccgacg actactaaag aaccggcacc caccacgcct aaagaaccag 120cccctactac gacaaaggag cctgcaccca caaccacgaa gagcgcaccc acaacaccaa 180aggagccggc ccctacgact cctaaagaac cagcccctac tacgacaaag gagcctgcac 240ccacaaccac gaagagcgca cccacaacac caaaggagcc ggcccctacg actcctaagg 300aacccaaacc ggcaccaacc actccgga 32817108PRTArtificial Sequence108 amino acids encoded by Lub3 DNA insert (9 KEPAPTT sequences between S373 and E482 in SEQ ID NO 15) 17Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala 1 5 10 15 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala 20 25 30 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala 35 40 45 Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro 50 55 60 Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala Pro 65 70 75 80 Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr 85 90 95 Thr Pro Lys Glu Pro Lys Pro Ala Pro Thr Thr Pro 100 105 183210DNAArtificial Sequencerecombinant PRG4-Lub4 cDNA construct. 18atggcatgga aaacacttcc catttacctg ttgttgctgc tgtctgtttt cgtgattcag 60caagtttcat ctcaagattt atcaagctgt gcagggagat gtggggaagg gtattctaga 120gatgccacct gcaactgtga ttataactgt caacactaca tggagtgctg ccctgatttc 180aagagagtct gcactgcgga gctttcctgt aaaggccgct gctttgagtc cttcgagaga 240gggagggagt gtgactgcga cgcccaatgt aagaagtatg acaagtgctg tcccgattat 300gagagtttct gtgcagaagt gcataatccc acatcaccac catcttcaaa gaaagcacct 360ccaccttcag gagcatctca aaccatcaaa tcaacaacca aacgttcacc caaaccacca 420aacaagaaga agactaagaa agttatagaa tcagaggaaa taacagaaga acattctgtt 480tctgaaaatc aagagtcctc ctccagtagc agttcaagta gttcgtcgtc gacaatttgg 540aaaatcaagt cttccaaaaa ttcagctgct aatagagaat tacagaagaa actcaaagta 600aaagataaca agaagaacag aactaaaaag aaacctaccc ccaaaccacc agttgtagat 660gaagctggaa gtggattgga caatggtgac ttcaaggtca caactcctga cacgtctacc 720acccaacaca ataaagtcag cacatctccc aagatcacaa cagcaaaacc aataaatccc 780agacccagtc ttccacctaa ttctgataca tctaaagaga cgtctttgac agtgaataaa 840gagacaacag ttgaaactaa agaaactact acaacaaata aacagacttc aactgatgga 900aaagagaaga ctacttccgc taaagagaca caaagtatag agaaaacatc tgctaaagat 960ttagcaccca catctaaagt gctggctaaa cctacaccca aagctgaaac tacaaccaaa 1020ggccctgctc tcaccactcc caaggagccc acgcccacca ctcccaagga gcctgcatct 1080accacaccca aagagcccac acctaccacc atcaagagcg cgcccacaac tccaaaagag 1140cccgcaccta ccacgacaaa gtcagctcct actacgccca aagagccagc gccgacgact 1200actaaagaac cggcacccac cacgcctaaa gaaccagccc ctactacgac aaaggagcct 1260gcacccacaa ccacgaagag cgcacccaca acaccaaagg agccggcccc tacgactcct 1320aaagaaccag cccctactac gacaaaggag cctgcaccca caaccacgaa gagcgcaccc 1380acaacaccaa aggagccggc ccctacgact cctaaagaac cagcccctac tacgacaaag 1440gagcctgcac ccacaaccac gaagagcgca cccacaacac caaaggagcc ggcccctacg 1500actcctaagg aacccaaacc ggcaccaacc actccggaaa cacctcctcc aaccacttca 1560gaggtctcta ctccaactac caccaaggag cctaccacta tccacaaaag ccctgatgaa 1620tcaactcctg agctttctgc agaacccaca ccaaaagctc ttgaaaacag tcccaaggaa 1680cctggtgtac ctacaactaa gacgccggcg gcgactaaac ctgaaatgac tacaacagct 1740aaagacaaga caacagaaag agacttacgt actacacctg aaactacaac tgctgcacct 1800aagatgacaa aagagacagc aactacaaca gaaaaaacta ccgaatccaa aataacagct 1860acaaccacac aagtaacatc taccacaact caagatacca caccattcaa aattactact 1920cttaaaacaa ctactcttgc acccaaagta actacaacaa aaaagacaat tactaccact 1980gagattatga acaaacctga agaaacagct aaaccaaaag acagagctac taattctaaa 2040gcgacaactc ctaaacctca aaagccaacc aaagcaccca aaaaacccac ttctaccaaa 2100aagccaaaaa caatgcctag agtgagaaaa ccaaagacga caccaactcc ccgcaagatg 2160acatcaacaa tgccagaatt gaaccctacc tcaagaatag cagaagccat gctccaaacc 2220accaccagac ctaaccaaac tccaaactcc aaactagttg aagtaaatcc aaagagtgaa 2280gatgcaggtg gtgctgaagg agaaacacct catatgcttc tcaggcccca tgtgttcatg 2340cctgaagtta ctcccgacat ggattactta ccgagagtac ccaatcaagg cattatcatc 2400aatcccatgc tttccgatga gaccaatata tgcaatggta agccagtaga tggactgact 2460actttgcgca atgggacatt agttgcattc cgaggtcatt atttctggat gctaagtcca 2520ttcagtccac catctccagc tcgcagaatt actgaagttt ggggtattcc ttcccccatt 2580gatactgttt ttactaggtg caactgtgaa ggaaaaactt tcttctttaa ggattctcag 2640tactggcgtt ttaccaatga tataaaagat gcagggtacc ccaaaccaat tttcaaagga 2700tttggaggac taactggaca aatagtggca gcgctttcaa cagctaaata taagaactgg 2760cctgaatctg tgtatttttt caagagaggt ggcagcattc agcagtatat ttataaacag 2820gaacctgtac agaagtgccc tggaagaagg cctgctctaa attatccagt gtatggagaa 2880atgacacagg ttaggagacg tcgctttgaa cgtgctatag gaccttctca aacacacacc 2940atcagaattc aatattcacc tgccagactg gcttatcaag acaaaggtgt ccttcataat 3000gaagttaaag tgagtatact gtggagagga cttccaaatg tggttacctc agctatatca 3060ctgcccaaca tcagaaaacc tgacggctat gattactatg ccttttctaa agatcaatac 3120tataacattg atgtgcctag tagaacagca agagcaatta ctactcgttc tgggcagacc 3180ttatccaaag tctggtacaa ctgtccttaa 3210191069PRTArtificial Sequenceamino acid sequence of entire PRG4-LUB4 protein. 19Met Ala Trp Lys Thr Leu Pro Ile Tyr Leu Leu Leu Leu Leu Ser Val 1 5 10

15 Phe Val Ile Gln Gln Val Ser Ser Gln Asp Leu Ser Ser Cys Ala Gly 20 25 30 Arg Cys Gly Glu Gly Tyr Ser Arg Asp Ala Thr Cys Asn Cys Asp Tyr 35 40 45 Asn Cys Gln His Tyr Met Glu Cys Cys Pro Asp Phe Lys Arg Val Cys 50 55 60 Thr Ala Glu Leu Ser Cys Lys Gly Arg Cys Phe Glu Ser Phe Glu Arg 65 70 75 80 Gly Arg Glu Cys Asp Cys Asp Ala Gln Cys Lys Lys Tyr Asp Lys Cys 85 90 95 Cys Pro Asp Tyr Glu Ser Phe Cys Ala Glu Val His Asn Pro Thr Ser 100 105 110 Pro Pro Ser Ser Lys Lys Ala Pro Pro Pro Ser Gly Ala Ser Gln Thr 115 120 125 Ile Lys Ser Thr Thr Lys Arg Ser Pro Lys Pro Pro Asn Lys Lys Lys 130 135 140 Thr Lys Lys Val Ile Glu Ser Glu Glu Ile Thr Glu Glu His Ser Val 145 150 155 160 Ser Glu Asn Gln Glu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 165 170 175 Ser Thr Ile Trp Lys Ile Lys Ser Ser Lys Asn Ser Ala Ala Asn Arg 180 185 190 Glu Leu Gln Lys Lys Leu Lys Val Lys Asp Asn Lys Lys Asn Arg Thr 195 200 205 Lys Lys Lys Pro Thr Pro Lys Pro Pro Val Val Asp Glu Ala Gly Ser 210 215 220 Gly Leu Asp Asn Gly Asp Phe Lys Val Thr Thr Pro Asp Thr Ser Thr 225 230 235 240 Thr Gln His Asn Lys Val Ser Thr Ser Pro Lys Ile Thr Thr Ala Lys 245 250 255 Pro Ile Asn Pro Arg Pro Ser Leu Pro Pro Asn Ser Asp Thr Ser Lys 260 265 270 Glu Thr Ser Leu Thr Val Asn Lys Glu Thr Thr Val Glu Thr Lys Glu 275 280 285 Thr Thr Thr Thr Asn Lys Gln Thr Ser Thr Asp Gly Lys Glu Lys Thr 290 295 300 Thr Ser Ala Lys Glu Thr Gln Ser Ile Glu Lys Thr Ser Ala Lys Asp 305 310 315 320 Leu Ala Pro Thr Ser Lys Val Leu Ala Lys Pro Thr Pro Lys Ala Glu 325 330 335 Thr Thr Thr Lys Gly Pro Ala Leu Thr Thr Pro Lys Glu Pro Thr Pro 340 345 350 Thr Thr Pro Lys Glu Pro Ala Ser Thr Thr Pro Lys Glu Pro Thr Pro 355 360 365 Thr Thr Ile Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr 370 375 380 Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 385 390 395 400 Thr Lys Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 405 410 415 Thr Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro 420 425 430 Lys Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr 435 440 445 Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys 450 455 460 Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys 465 470 475 480 Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu 485 490 495 Pro Ala Pro Thr Thr Pro Lys Glu Pro Lys Pro Ala Pro Thr Thr Pro 500 505 510 Glu Thr Pro Pro Pro Thr Thr Ser Glu Val Ser Thr Pro Thr Thr Thr 515 520 525 Lys Glu Pro Thr Thr Ile His Lys Ser Pro Asp Glu Ser Thr Pro Glu 530 535 540 Leu Ser Ala Glu Pro Thr Pro Lys Ala Leu Glu Asn Ser Pro Lys Glu 545 550 555 560 Pro Gly Val Pro Thr Thr Lys Thr Pro Ala Ala Thr Lys Pro Glu Met 565 570 575 Thr Thr Thr Ala Lys Asp Lys Thr Thr Glu Arg Asp Leu Arg Thr Thr 580 585 590 Pro Glu Thr Thr Thr Ala Ala Pro Lys Met Thr Lys Glu Thr Ala Thr 595 600 605 Thr Thr Glu Lys Thr Thr Glu Ser Lys Ile Thr Ala Thr Thr Thr Gln 610 615 620 Val Thr Ser Thr Thr Thr Gln Asp Thr Thr Pro Phe Lys Ile Thr Thr 625 630 635 640 Leu Lys Thr Thr Thr Leu Ala Pro Lys Val Thr Thr Thr Lys Lys Thr 645 650 655 Ile Thr Thr Thr Glu Ile Met Asn Lys Pro Glu Glu Thr Ala Lys Pro 660 665 670 Lys Asp Arg Ala Thr Asn Ser Lys Ala Thr Thr Pro Lys Pro Gln Lys 675 680 685 Pro Thr Lys Ala Pro Lys Lys Pro Thr Ser Thr Lys Lys Pro Lys Thr 690 695 700 Met Pro Arg Val Arg Lys Pro Lys Thr Thr Pro Thr Pro Arg Lys Met 705 710 715 720 Thr Ser Thr Met Pro Glu Leu Asn Pro Thr Ser Arg Ile Ala Glu Ala 725 730 735 Met Leu Gln Thr Thr Thr Arg Pro Asn Gln Thr Pro Asn Ser Lys Leu 740 745 750 Val Glu Val Asn Pro Lys Ser Glu Asp Ala Gly Gly Ala Glu Gly Glu 755 760 765 Thr Pro His Met Leu Leu Arg Pro His Val Phe Met Pro Glu Val Thr 770 775 780 Pro Asp Met Asp Tyr Leu Pro Arg Val Pro Asn Gln Gly Ile Ile Ile 785 790 795 800 Asn Pro Met Leu Ser Asp Glu Thr Asn Ile Cys Asn Gly Lys Pro Val 805 810 815 Asp Gly Leu Thr Thr Leu Arg Asn Gly Thr Leu Val Ala Phe Arg Gly 820 825 830 His Tyr Phe Trp Met Leu Ser Pro Phe Ser Pro Pro Ser Pro Ala Arg 835 840 845 Arg Ile Thr Glu Val Trp Gly Ile Pro Ser Pro Ile Asp Thr Val Phe 850 855 860 Thr Arg Cys Asn Cys Glu Gly Lys Thr Phe Phe Phe Lys Asp Ser Gln 865 870 875 880 Tyr Trp Arg Phe Thr Asn Asp Ile Lys Asp Ala Gly Tyr Pro Lys Pro 885 890 895 Ile Phe Lys Gly Phe Gly Gly Leu Thr Gly Gln Ile Val Ala Ala Leu 900 905 910 Ser Thr Ala Lys Tyr Lys Asn Trp Pro Glu Ser Val Tyr Phe Phe Lys 915 920 925 Arg Gly Gly Ser Ile Gln Gln Tyr Ile Tyr Lys Gln Glu Pro Val Gln 930 935 940 Lys Cys Pro Gly Arg Arg Pro Ala Leu Asn Tyr Pro Val Tyr Gly Glu 945 950 955 960 Met Thr Gln Val Arg Arg Arg Arg Phe Glu Arg Ala Ile Gly Pro Ser 965 970 975 Gln Thr His Thr Ile Arg Ile Gln Tyr Ser Pro Ala Arg Leu Ala Tyr 980 985 990 Gln Asp Lys Gly Val Leu His Asn Glu Val Lys Val Ser Ile Leu Trp 995 1000 1005 Arg Gly Leu Pro Asn Val Val Thr Ser Ala Ile Ser Leu Pro Asn 1010 1015 1020 Ile Arg Lys Pro Asp Gly Tyr Asp Tyr Tyr Ala Phe Ser Lys Asp 1025 1030 1035 Gln Tyr Tyr Asn Ile Asp Val Pro Ser Arg Thr Ala Arg Ala Ile 1040 1045 1050 Thr Thr Arg Ser Gly Gln Thr Leu Ser Lys Val Trp Tyr Asn Cys 1055 1060 1065 Pro 20421DNAArtificial SequenceLub4 DNA insert from cDNA cassette-1 and three synthetic cDNA cassette-2 sequences. 20gcgcgcccac aactccaaaa gagcccgcac ctaccacgac aaagtcagct cctactacgc 60ccaaagagcc agcgccgacg actactaaag aaccggcacc caccacgcct aaagaaccag 120cccctactac gacaaaggag cctgcaccca caaccacgaa gagcgcaccc acaacaccaa 180aggagccggc ccctacgact cctaaagaac cagcccctac tacgacaaag gagcctgcac 240ccacaaccac gaagagcgca cccacaacac caaaggagcc ggcccctacg actcctaaag 300aaccagcccc tactacgaca aaggagcctg cacccacaac cacgaagagc gcacccacaa 360caccaaagga gccggcccct acgactccta aggaacccaa accggcacca accactccgg 420a 42121139PRTArtificial Sequence139 amino acids encoded by Lub4 DNA insert (12 KEPAPTT sequences between S373 and E513 in SEQ ID NO 19) 21Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala 1 5 10 15 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala 20 25 30 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala 35 40 45 Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro 50 55 60 Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala Pro 65 70 75 80 Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr 85 90 95 Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala Pro Thr 100 105 110 Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 115 120 125 Pro Lys Glu Pro Lys Pro Ala Pro Thr Thr Pro 130 135 223303DNAArtificial SequenceRecombinant PRG4-Lub5 cDNA construct 22atggcatgga aaacacttcc catttacctg ttgttgctgc tgtctgtttt cgtgattcag 60caagtttcat ctcaagattt atcaagctgt gcagggagat gtggggaagg gtattctaga 120gatgccacct gcaactgtga ttataactgt caacactaca tggagtgctg ccctgatttc 180aagagagtct gcactgcgga gctttcctgt aaaggccgct gctttgagtc cttcgagaga 240gggagggagt gtgactgcga cgcccaatgt aagaagtatg acaagtgctg tcccgattat 300gagagtttct gtgcagaagt gcataatccc acatcaccac catcttcaaa gaaagcacct 360ccaccttcag gagcatctca aaccatcaaa tcaacaacca aacgttcacc caaaccacca 420aacaagaaga agactaagaa agttatagaa tcagaggaaa taacagaaga acattctgtt 480tctgaaaatc aagagtcctc ctccagtagc agttcaagta gttcgtcgtc gacaatttgg 540aaaatcaagt cttccaaaaa ttcagctgct aatagagaat tacagaagaa actcaaagta 600aaagataaca agaagaacag aactaaaaag aaacctaccc ccaaaccacc agttgtagat 660gaagctggaa gtggattgga caatggtgac ttcaaggtca caactcctga cacgtctacc 720acccaacaca ataaagtcag cacatctccc aagatcacaa cagcaaaacc aataaatccc 780agacccagtc ttccacctaa ttctgataca tctaaagaga cgtctttgac agtgaataaa 840gagacaacag ttgaaactaa agaaactact acaacaaata aacagacttc aactgatgga 900aaagagaaga ctacttccgc taaagagaca caaagtatag agaaaacatc tgctaaagat 960ttagcaccca catctaaagt gctggctaaa cctacaccca aagctgaaac tacaaccaaa 1020ggccctgctc tcaccactcc caaggagccc acgcccacca ctcccaagga gcctgcatct 1080accacaccca aagagcccac acctaccacc atcaagagcg cgcccacaac tccaaaagag 1140cccgcaccta ccacgacaaa gtcagctcct actacgccca aagagccagc gccgacgact 1200actaaagaac cggcacccac cacgcctaaa gaaccagccc ctactacgac aaaggagcct 1260gcacccacaa ccacgaagag cgcacccaca acaccaaagg agccggcccc tacgactcct 1320aaagaaccag cccctactac gacaaaggag cctgcaccca caaccacgaa gagcgcaccc 1380acaacaccaa aggagccggc ccctacgact cctaaagaac cagcccctac tacgacaaag 1440gagcctgcac ccacaaccac gaagagcgca cccacaacac caaaggagcc ggcccctacg 1500actcctaaag aaccagcccc tactacgaca aaggagcctg cacccacaac cacgaagagc 1560gcacccacaa caccaaagga gccggcccct acgactccta aggaacccaa accggcacca 1620accactccgg aaacacctcc tccaaccact tcagaggtct ctactccaac taccaccaag 1680gagcctacca ctatccacaa aagccctgat gaatcaactc ctgagctttc tgcagaaccc 1740acaccaaaag ctcttgaaaa cagtcccaag gaacctggtg tacctacaac taagacgccg 1800gcggcgacta aacctgaaat gactacaaca gctaaagaca agacaacaga aagagactta 1860cgtactacac ctgaaactac aactgctgca cctaagatga caaaagagac agcaactaca 1920acagaaaaaa ctaccgaatc caaaataaca gctacaacca cacaagtaac atctaccaca 1980actcaagata ccacaccatt caaaattact actcttaaaa caactactct tgcacccaaa 2040gtaactacaa caaaaaagac aattactacc actgagatta tgaacaaacc tgaagaaaca 2100gctaaaccaa aagacagagc tactaattct aaagcgacaa ctcctaaacc tcaaaagcca 2160accaaagcac ccaaaaaacc cacttctacc aaaaagccaa aaacaatgcc tagagtgaga 2220aaaccaaaga cgacaccaac tccccgcaag atgacatcaa caatgccaga attgaaccct 2280acctcaagaa tagcagaagc catgctccaa accaccacca gacctaacca aactccaaac 2340tccaaactag ttgaagtaaa tccaaagagt gaagatgcag gtggtgctga aggagaaaca 2400cctcatatgc ttctcaggcc ccatgtgttc atgcctgaag ttactcccga catggattac 2460ttaccgagag tacccaatca aggcattatc atcaatccca tgctttccga tgagaccaat 2520atatgcaatg gtaagccagt agatggactg actactttgc gcaatgggac attagttgca 2580ttccgaggtc attatttctg gatgctaagt ccattcagtc caccatctcc agctcgcaga 2640attactgaag tttggggtat tccttccccc attgatactg tttttactag gtgcaactgt 2700gaaggaaaaa ctttcttctt taaggattct cagtactggc gttttaccaa tgatataaaa 2760gatgcagggt accccaaacc aattttcaaa ggatttggag gactaactgg acaaatagtg 2820gcagcgcttt caacagctaa atataagaac tggcctgaat ctgtgtattt tttcaagaga 2880ggtggcagca ttcagcagta tatttataaa caggaacctg tacagaagtg ccctggaaga 2940aggcctgctc taaattatcc agtgtatgga gaaatgacac aggttaggag acgtcgcttt 3000gaacgtgcta taggaccttc tcaaacacac accatcagaa ttcaatattc acctgccaga 3060ctggcttatc aagacaaagg tgtccttcat aatgaagtta aagtgagtat actgtggaga 3120ggacttccaa atgtggttac ctcagctata tcactgccca acatcagaaa acctgacggc 3180tatgattact atgccttttc taaagatcaa tactataaca ttgatgtgcc tagtagaaca 3240gcaagagcaa ttactactcg ttctgggcag accttatcca aagtctggta caactgtcct 3300taa 3303231100PRTArtificial SequenceAmino acid sequence of entire PRG4-LUB5 protein. 23Met Ala Trp Lys Thr Leu Pro Ile Tyr Leu Leu Leu Leu Leu Ser Val 1 5 10 15 Phe Val Ile Gln Gln Val Ser Ser Gln Asp Leu Ser Ser Cys Ala Gly 20 25 30 Arg Cys Gly Glu Gly Tyr Ser Arg Asp Ala Thr Cys Asn Cys Asp Tyr 35 40 45 Asn Cys Gln His Tyr Met Glu Cys Cys Pro Asp Phe Lys Arg Val Cys 50 55 60 Thr Ala Glu Leu Ser Cys Lys Gly Arg Cys Phe Glu Ser Phe Glu Arg 65 70 75 80 Gly Arg Glu Cys Asp Cys Asp Ala Gln Cys Lys Lys Tyr Asp Lys Cys 85 90 95 Cys Pro Asp Tyr Glu Ser Phe Cys Ala Glu Val His Asn Pro Thr Ser 100 105 110 Pro Pro Ser Ser Lys Lys Ala Pro Pro Pro Ser Gly Ala Ser Gln Thr 115 120 125 Ile Lys Ser Thr Thr Lys Arg Ser Pro Lys Pro Pro Asn Lys Lys Lys 130 135 140 Thr Lys Lys Val Ile Glu Ser Glu Glu Ile Thr Glu Glu His Ser Val 145 150 155 160 Ser Glu Asn Gln Glu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 165 170 175 Ser Thr Ile Trp Lys Ile Lys Ser Ser Lys Asn Ser Ala Ala Asn Arg 180 185 190 Glu Leu Gln Lys Lys Leu Lys Val Lys Asp Asn Lys Lys Asn Arg Thr 195 200 205 Lys Lys Lys Pro Thr Pro Lys Pro Pro Val Val Asp Glu Ala Gly Ser 210 215 220 Gly Leu Asp Asn Gly Asp Phe Lys Val Thr Thr Pro Asp Thr Ser Thr 225 230 235 240 Thr Gln His Asn Lys Val Ser Thr Ser Pro Lys Ile Thr Thr Ala Lys 245 250 255 Pro Ile Asn Pro Arg Pro Ser Leu Pro Pro Asn Ser Asp Thr Ser Lys 260 265 270 Glu Thr Ser Leu Thr Val Asn Lys Glu Thr Thr Val Glu Thr Lys Glu 275 280 285 Thr Thr Thr Thr Asn Lys Gln Thr Ser Thr Asp Gly Lys Glu Lys Thr 290 295 300 Thr Ser Ala Lys Glu Thr Gln Ser Ile Glu Lys Thr Ser Ala Lys Asp 305 310 315 320 Leu Ala Pro Thr Ser Lys Val Leu Ala Lys Pro Thr Pro Lys Ala Glu 325 330 335 Thr Thr Thr Lys Gly Pro Ala Leu Thr Thr Pro Lys Glu Pro Thr Pro 340 345 350 Thr Thr Pro Lys Glu Pro Ala Ser Thr Thr Pro Lys Glu Pro Thr Pro 355 360 365 Thr Thr Ile Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr 370 375 380 Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 385 390 395 400 Thr Lys Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 405 410 415 Thr Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro 420 425 430 Lys Glu Pro Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr 435 440 445 Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys 450 455 460 Glu Pro Ala Pro Thr

Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys 465 470 475 480 Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu 485 490 495 Pro Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu 500 505 510 Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro 515 520 525 Ala Pro Thr Thr Pro Lys Glu Pro Lys Pro Ala Pro Thr Thr Pro Glu 530 535 540 Thr Pro Pro Pro Thr Thr Ser Glu Val Ser Thr Pro Thr Thr Thr Lys 545 550 555 560 Glu Pro Thr Thr Ile His Lys Ser Pro Asp Glu Ser Thr Pro Glu Leu 565 570 575 Ser Ala Glu Pro Thr Pro Lys Ala Leu Glu Asn Ser Pro Lys Glu Pro 580 585 590 Gly Val Pro Thr Thr Lys Thr Pro Ala Ala Thr Lys Pro Glu Met Thr 595 600 605 Thr Thr Ala Lys Asp Lys Thr Thr Glu Arg Asp Leu Arg Thr Thr Pro 610 615 620 Glu Thr Thr Thr Ala Ala Pro Lys Met Thr Lys Glu Thr Ala Thr Thr 625 630 635 640 Thr Glu Lys Thr Thr Glu Ser Lys Ile Thr Ala Thr Thr Thr Gln Val 645 650 655 Thr Ser Thr Thr Thr Gln Asp Thr Thr Pro Phe Lys Ile Thr Thr Leu 660 665 670 Lys Thr Thr Thr Leu Ala Pro Lys Val Thr Thr Thr Lys Lys Thr Ile 675 680 685 Thr Thr Thr Glu Ile Met Asn Lys Pro Glu Glu Thr Ala Lys Pro Lys 690 695 700 Asp Arg Ala Thr Asn Ser Lys Ala Thr Thr Pro Lys Pro Gln Lys Pro 705 710 715 720 Thr Lys Ala Pro Lys Lys Pro Thr Ser Thr Lys Lys Pro Lys Thr Met 725 730 735 Pro Arg Val Arg Lys Pro Lys Thr Thr Pro Thr Pro Arg Lys Met Thr 740 745 750 Ser Thr Met Pro Glu Leu Asn Pro Thr Ser Arg Ile Ala Glu Ala Met 755 760 765 Leu Gln Thr Thr Thr Arg Pro Asn Gln Thr Pro Asn Ser Lys Leu Val 770 775 780 Glu Val Asn Pro Lys Ser Glu Asp Ala Gly Gly Ala Glu Gly Glu Thr 785 790 795 800 Pro His Met Leu Leu Arg Pro His Val Phe Met Pro Glu Val Thr Pro 805 810 815 Asp Met Asp Tyr Leu Pro Arg Val Pro Asn Gln Gly Ile Ile Ile Asn 820 825 830 Pro Met Leu Ser Asp Glu Thr Asn Ile Cys Asn Gly Lys Pro Val Asp 835 840 845 Gly Leu Thr Thr Leu Arg Asn Gly Thr Leu Val Ala Phe Arg Gly His 850 855 860 Tyr Phe Trp Met Leu Ser Pro Phe Ser Pro Pro Ser Pro Ala Arg Arg 865 870 875 880 Ile Thr Glu Val Trp Gly Ile Pro Ser Pro Ile Asp Thr Val Phe Thr 885 890 895 Arg Cys Asn Cys Glu Gly Lys Thr Phe Phe Phe Lys Asp Ser Gln Tyr 900 905 910 Trp Arg Phe Thr Asn Asp Ile Lys Asp Ala Gly Tyr Pro Lys Pro Ile 915 920 925 Phe Lys Gly Phe Gly Gly Leu Thr Gly Gln Ile Val Ala Ala Leu Ser 930 935 940 Thr Ala Lys Tyr Lys Asn Trp Pro Glu Ser Val Tyr Phe Phe Lys Arg 945 950 955 960 Gly Gly Ser Ile Gln Gln Tyr Ile Tyr Lys Gln Glu Pro Val Gln Lys 965 970 975 Cys Pro Gly Arg Arg Pro Ala Leu Asn Tyr Pro Val Tyr Gly Glu Met 980 985 990 Thr Gln Val Arg Arg Arg Arg Phe Glu Arg Ala Ile Gly Pro Ser Gln 995 1000 1005 Thr His Thr Ile Arg Ile Gln Tyr Ser Pro Ala Arg Leu Ala Tyr 1010 1015 1020 Gln Asp Lys Gly Val Leu His Asn Glu Val Lys Val Ser Ile Leu 1025 1030 1035 Trp Arg Gly Leu Pro Asn Val Val Thr Ser Ala Ile Ser Leu Pro 1040 1045 1050 Asn Ile Arg Lys Pro Asp Gly Tyr Asp Tyr Tyr Ala Phe Ser Lys 1055 1060 1065 Asp Gln Tyr Tyr Asn Ile Asp Val Pro Ser Arg Thr Ala Arg Ala 1070 1075 1080 Ile Thr Thr Arg Ser Gly Gln Thr Leu Ser Lys Val Trp Tyr Asn 1085 1090 1095 Cys Pro 1100 24514DNAArtificial SequenceLub5 DNA insert from cDNA cassette-1 and four synthetic cDNA cassette-2 sequences 24gcgcgcccac aactccaaaa gagcccgcac ctaccacgac aaagtcagct cctactacgc 60ccaaagagcc agcgccgacg actactaaag aaccggcacc caccacgcct aaagaaccag 120cccctactac gacaaaggag cctgcaccca caaccacgaa gagcgcaccc acaacaccaa 180aggagccggc ccctacgact cctaaagaac cagcccctac tacgacaaag gagcctgcac 240ccacaaccac gaagagcgca cccacaacac caaaggagcc ggcccctacg actcctaaag 300aaccagcccc tactacgaca aaggagcctg cacccacaac cacgaagagc gcacccacaa 360caccaaagga gccggcccct acgactccta aagaaccagc ccctactacg acaaaggagc 420ctgcacccac aaccacgaag agcgcaccca caacaccaaa ggagccggcc cctacgactc 480ctaaggaacc caaaccggca ccaaccactc cgga 51425170PRTArtificial Sequence170 amino acids encoded by Lub5 DNA insert (15 KEPAPTT sequences between S373 and E544 in SEQ ID NO 23) 25Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala 1 5 10 15 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala 20 25 30 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala 35 40 45 Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro 50 55 60 Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala Pro 65 70 75 80 Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr 85 90 95 Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala Pro Thr 100 105 110 Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr 115 120 125 Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala Pro Thr Thr 130 135 140 Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Pro 145 150 155 160 Lys Glu Pro Lys Pro Ala Pro Thr Thr Pro 165 170 2645PRTArtificial Sequenceamino acid sequence "APTTPKEPAPTTTKSAPTTPKEPAPTTT KEPAPTTPKEPAPTTTK" (45 amino acids) in preferred PRG4-LUBN protein 26Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala 1 5 10 15 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala 20 25 30 Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Thr Lys 35 40 45 2731PRTArtificial Sequenceamino acid sequence "KEPAPTTTKEPAPTTTKSAPTTPKEPAPTTP" (31 amino acids) repeated N-1 times in preferred PRG4-LUBN protein 27Lys Glu Pro Ala Pro Thr Thr Thr Lys Glu Pro Ala Pro Thr Thr Thr 1 5 10 15 Lys Ser Ala Pro Thr Thr Pro Lys Glu Pro Ala Pro Thr Thr Pro 20 25 30 2822PRTArtificial SequenceAmino acid sequence "EPAPTTTKSAPTTPKEPAPTTP" (22 amino acids) joining SEQ ID NO 26 to (N-2) repeats of SEQ ID NO 27 in preferred PRG4-LUBN protein where N = 3 or more. 28Glu Pro Ala Pro Thr Thr Thr Lys Ser Ala Pro Thr Thr Pro Lys Glu 1 5 10 15 Pro Ala Pro Thr Thr Pro 20 2910PRTArtificial SequenceAmino acid sequence "KEPKPAPTTP" (10 amino acids) in preferred PRG4-LUBN protein where N = 2 or more. 29Lys Glu Pro Lys Pro Ala Pro Thr Thr Pro 1 5 10

Claims

1.-10. (canceled)

11. An isolated protein comprising the amino acid sequence of SEQ ID NO: 15.

12-32. (canceled)

33. The protein of claim 11, wherein the protein is O-linked to .beta.-(1-3)-Gal-GalNac.

34. A composition comprising a therapeutically effective amount of a protein of claim 33 in a pharmaceutically acceptable carrier.

35. The composition of claim 34, additionally comprising hyaluronan or hylan.

36. The composition of claim 34, wherein the composition is formulated for injection.

37. The composition of claim 34, wherein the composition is encapsulated.

38. The composition of claim 37, wherein the composition is encapsulated in an implantable drug delivery matrix.

39. The composition of claim 34, wherein the composition further comprises a local anesthetic.

40. The composition of claim 34, wherein the composition further comprises a cytokine.

41. The composition of claim 34, wherein the composition further comprises a lymphokine.

42. The composition of claim 34, wherein the composition further comprises a thrombolytic factor.

43. The composition of claim 34, wherein the composition further comprises an antithrombolytic factor.

44. The composition of claim 34, wherein the composition further comprises an anti-inflammatory agent.

45. The composition of claim 34, wherein the composition further comprises an antibiotic.

46. The composition of claim 34, wherein the composition further comprises a growth factor.