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Regulation of Hyaluronidase Activity by Alternative mRNA Splicing

2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês

10.1074/jbc.m203821200

ISSN

1083-351X

Autores

Vinata B. Lokeshwar, Grethchen L. Schroeder, Robert I. Carey, Mark S. Soloway, Naoko Iida,

Tópico(s)

Glycosylation and Glycoproteins Research

Resumo

Hyaluronidase is a hyaluronic acid-degrading endoglycosidase that is present in many toxins and the levels of which are elevated in cancer. Increased concentration of HYAL1-type hyaluronidase correlates with tumor progression and is a marker for grade (G) 2 or 3 bladder cancer. Using bladder tissues and cells, prostate cancer cells, and kidney tissues and performing reverse transcription-PCR, cDNA cloning, DNA sequencing, and in vitro translation, we identified splice variants of HYAL1 and HYAL3. HYAL1v1 variant lacks a 30-amino acid (aa) sequence (301–330) present in HYAL1 protein. HYAL1v1, HYAL1v2 (aa 183–435 present in HYAL1 wild type), HYAL1v3 (aa 1–207), HYAL1v4 (aa 260–435), and HYAL1v5 (aa 340–435) are enzymatically inactive and are expressed in normal tissues/cells and G1 bladder tumor tissues. However, HYAL1 wild type is expressed in G2/G3 tumors and in invasive tumor cells. Stable transfection and HYAL1v1-specific antibody confirmed that the HYAL1 sequence from aa 301 to 330 is critical for hyaluronidase activity. All tumor cells and tissues mainly express HYAL3 variants. HYAL3v1 lacks a 30-aa sequence (299–328) present in HYAL3 protein, that is homologous to the 30-aa HYAL1 sequence. HYAL3v1, HYAL3v2 (aa 251–417 present in HYAL3 wild type), and HYAL3v3 (aa 251–417, but lacking aa 299–328), are enzymatically inactive. Although splicing of a single independent exon generates HYAL1v1 and HYAL3v1, internal exon splicing generates the other HYAL1/HYAL3 variants. These results demonstrate that alternative mRNA splicing controls cellular expression of enzymatically active hyaluronidase and may explain the elevated hyaluronidase levels in bladder/prostate cancer. Hyaluronidase is a hyaluronic acid-degrading endoglycosidase that is present in many toxins and the levels of which are elevated in cancer. Increased concentration of HYAL1-type hyaluronidase correlates with tumor progression and is a marker for grade (G) 2 or 3 bladder cancer. Using bladder tissues and cells, prostate cancer cells, and kidney tissues and performing reverse transcription-PCR, cDNA cloning, DNA sequencing, and in vitro translation, we identified splice variants of HYAL1 and HYAL3. HYAL1v1 variant lacks a 30-amino acid (aa) sequence (301–330) present in HYAL1 protein. HYAL1v1, HYAL1v2 (aa 183–435 present in HYAL1 wild type), HYAL1v3 (aa 1–207), HYAL1v4 (aa 260–435), and HYAL1v5 (aa 340–435) are enzymatically inactive and are expressed in normal tissues/cells and G1 bladder tumor tissues. However, HYAL1 wild type is expressed in G2/G3 tumors and in invasive tumor cells. Stable transfection and HYAL1v1-specific antibody confirmed that the HYAL1 sequence from aa 301 to 330 is critical for hyaluronidase activity. All tumor cells and tissues mainly express HYAL3 variants. HYAL3v1 lacks a 30-aa sequence (299–328) present in HYAL3 protein, that is homologous to the 30-aa HYAL1 sequence. HYAL3v1, HYAL3v2 (aa 251–417 present in HYAL3 wild type), and HYAL3v3 (aa 251–417, but lacking aa 299–328), are enzymatically inactive. Although splicing of a single independent exon generates HYAL1v1 and HYAL3v1, internal exon splicing generates the other HYAL1/HYAL3 variants. These results demonstrate that alternative mRNA splicing controls cellular expression of enzymatically active hyaluronidase and may explain the elevated hyaluronidase levels in bladder/prostate cancer. hyaluronidase amino acid(s) hyaluronic acid no evidence of disease bladder tumor reverse transcription enzyme-linked immunosorbent assay triose phosphate isomerase Hyaluronidases (HAases)1are a family of enzymes that are crucial for the spread of bacterial infections, toxins present in various venoms, and possibly, cancer progression (1Kreil G. Protein Sci. 1995; 4: 1666-1669Crossref PubMed Scopus (306) Google Scholar, 2Roden L. Campbell P. Fraser J.R.E. Laurent T.C. Petroff H. Thompson J.N. CIBA Found. Symp. 1989; 143: 60-86PubMed Google Scholar, 3Tu A.T. Hendon R.R. Comp. Biochem. Physiol. B. Comp. Biochem. 1983; 76: 377-383Crossref PubMed Scopus (23) Google Scholar, 4Gmachl M. Kreil G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3569-3573Crossref PubMed Scopus (177) Google Scholar, 5Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2589) Google Scholar, 6Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1178) Google Scholar). In humans, six HAase genes have been identified. These genes occur in clusters of three at two chromosomal locations (Ref. 7Csoka A.B. Frost G.I. Stern R. Matrix Biol. 2001; 20: 499-508Crossref PubMed Scopus (474) Google Scholar and human genome blast search). HYAL1, HYAL2, and HYAL3 occur on chromosome 3p21.3, and PH20, HYAL4, and HYALP1 occur on chromosome 7q31.3. With the possible exception of HYAL4 and HYALP1, all other HAases degrade hyaluronic acid (HA) (7Csoka A.B. Frost G.I. Stern R. Matrix Biol. 2001; 20: 499-508Crossref PubMed Scopus (474) Google Scholar).HA is a nonsulfated glycosaminoglycan made up of repeating disaccharide units, d-glucuronic acid, andN-acetyl-d-glucosamine. HA is present in body fluids, tissues, and the extracellular matrix (8Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2049) Google Scholar, 9Delpech B. Girard N. Bertrand P. Chauzy C. Delpech A. J. Intern. Med. 1997; 242: 41-48Crossref PubMed Scopus (141) Google Scholar, 10Tammi M.I. Day A.J. Turely E.A. J. Biol. Chem. 2002; 277: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). It keeps tissues hydrated and maintains osmotic balance and cartilage integrity (8Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2049) Google Scholar, 10Tammi M.I. Day A.J. Turely E.A. J. Biol. Chem. 2002; 277: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). HA also actively regulates cell adhesion, migration, and proliferation by interacting with specific cell surface receptors such as CD44 and RHAMM (11Turley E.A. Noble P.W. Bourguignon L.Y.W. J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar). The concentrations of HA are elevated in several inflammatory diseases and various carcinomas (e.g. bladder, prostate, breast, lung, colon, and so forth; Refs. 9Delpech B. Girard N. Bertrand P. Chauzy C. Delpech A. J. Intern. Med. 1997; 242: 41-48Crossref PubMed Scopus (141) Google Scholar and 12Toole B.P. Wight T.N. Tammi M.I. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 13Ropponen K. Tammi M.I. Parkkinen J. Eskelinen M.J. Tammi R.H. Lipponen P.R. Argen U.M. Alhava E.M. Kosma V.M. Cancer Res. 1998; 58: 342-347PubMed Google Scholar, 14Setala L.P. Tammi M.I. Tammi R.H. Eskelinen M.J. Lipponen P.R. Argen U.M. Alhava E.M. Kosma V.M. Br. J. Cancer. 1999; 79: 1133-1138Crossref PubMed Scopus (132) Google Scholar, 15Auvinen P. Tammi R.H. Parkkinen J. Tammi M.I. Argen U.M. Johansson R. Hirvikoski P. Eskelinen M.J. Kosma V.M. Am. J. Pathol. 2000; 156: 529-536Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 16Pirinen R. Tammi R. Tammi M. Hirvikoski P. Parkkinen J.J. Johansson R. Bohm J. Hollmen S. Kosma V.M. Int. J. Cancer. 2001; 95: 12-17Crossref PubMed Scopus (129) Google Scholar, 17Lokeshwar V.B. Rubinowicz D. Schroeder G.L. Forgacs E. Minna J.D. Block N.L. Nadji M. Lokeshwar B.L. J. Biol. Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 18Lokeshwar V.B. Obek C. Pham H.T. Wei D. Young M.J. Duncan R.C. Soloway M.S. Block N.L. J. Urol. 2000; 163: 348-356Crossref PubMed Scopus (173) Google Scholar, 19Lokeshwar V.B. Block N.L. Urol. Clin. North Am. 2000; 27: 53-60Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). For example, we have shown that urinary HA concentration is a highly sensitive and specific marker for detecting bladder cancer, regardless of its grade (18Lokeshwar V.B. Obek C. Pham H.T. Wei D. Young M.J. Duncan R.C. Soloway M.S. Block N.L. J. Urol. 2000; 163: 348-356Crossref PubMed Scopus (173) Google Scholar, 19Lokeshwar V.B. Block N.L. Urol. Clin. North Am. 2000; 27: 53-60Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In tumor tissues, HA may promote tumor growth and metastasis probably by actively supporting tumor cell migration and offering protection against immune surveillance (20Liu N. Lapcevich R.K. Underhill C.B. Han Z. Gao F. Swartz G. Plum S.M. Zhang L. Gree S.J. Cancer Res. 2001; 61: 1022-1028PubMed Google Scholar, 21Hayen W. Goebeler M. Kumar S. Riessen R. Nehls V. J. Cell Sci. 1999; 112: 2241-2251Crossref PubMed Google Scholar, 22Hobarth K. Maier U. Marberger M. Eur. Urol. 1992; 21: 206-210Crossref PubMed Scopus (63) Google Scholar). Small fragments of HA, generated by HAases, stimulate angiogenesis (23Slevin M. Krupinski J. Kumar S. Gaffney J. Lab. Invest. 1998; 78: 987-1003PubMed Google Scholar, 24Trochon V. Mabilat-Pragnon C. Bertrand P. Legrand Y. Soria J. Soria C. Delpech B. Lu H. FEBS Lett. 1997; 418: 6-10Crossref PubMed Scopus (45) Google Scholar, 25Rooney P. Kumar S. Ponting J. Wang M. Int. J. Cancer. 1995; 60: 632-636Crossref PubMed Scopus (258) Google Scholar). We recently showed that HA fragments of ∼10–15 disaccharide units stimulate endothelial cell proliferation by acting through cell surface HA receptor, RHAMM, and activating the mitogen-activated protein kinase pathway (26Lokeshwar V.B. Selzer M.G. J. Biol. Chem. 2000; 265: 27641-27649Abstract Full Text Full Text PDF Scopus (176) Google Scholar). We have also shown that elevated levels of HYAL1-type HAase coincide with the presence of angiogenic HA fragments in prostate tumor tissues and in the urine of bladder cancer patients (17Lokeshwar V.B. Rubinowicz D. Schroeder G.L. Forgacs E. Minna J.D. Block N.L. Nadji M. Lokeshwar B.L. J. Biol. Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 27Lokeshwar V.B. Obek C. Soloway M.S. Block N.L. Cancer Res. 1997; 57 (; Correction (1998) Cancer Res.58, 3191): 773-777PubMed Google Scholar).Among the six HAases, HYAL1, HYAL2, and PH20 are well characterized. HYAL1 type HAase was originally purified from human plasma and urine (28Frost G.I. Csoka T.B. Wong T. Stern R. Biochem. Biophys. Res. Commun. 1997; 236: 10-15Crossref PubMed Scopus (208) Google Scholar, 29Csoka T.B. Frost G.I. Wong T. Stern R. FEBS Lett. 1997; 417: 307-310Crossref PubMed Scopus (96) Google Scholar). However, we have shown that HYAL1 is the major tumor-derived HAase expressed in bladder and prostate cancer tissues (17Lokeshwar V.B. Rubinowicz D. Schroeder G.L. Forgacs E. Minna J.D. Block N.L. Nadji M. Lokeshwar B.L. J. Biol. Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 30Lokeshwar V.B. Young M.J. Gouddarzi G. Iida N. Yudin A.I. Cherr G.N. Selzer M.G. Cancer Res. 1999; 59: 4464-4470PubMed Google Scholar). It has a optimum pH range of 4.0–4.3, and the enzyme is 50–80% active at pH 4.5 (17Lokeshwar V.B. Rubinowicz D. Schroeder G.L. Forgacs E. Minna J.D. Block N.L. Nadji M. Lokeshwar B.L. J. Biol. Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Triggs-Raine et al. (31Triggs-Raine B. Salo T.J. Zhang H. Wicklow B.A. Natowicz M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6296-6300Crossref PubMed Scopus (165) Google Scholar) have shown that a lack of functional HYAL1 results in a disorder called mucopolysaccharidosis IX. In this study the authors identified that aa Glu268 is crucial for HYAL1 activity. HYAL2 was originally designated as the lysosomal HAase, and it cleaves high molecular mass HA into ∼20-kDa HA fragments (32Lepperdinger G. Mullegger J. Kreil G. Matrix Biol. 2001; 20: 509-514Crossref PubMed Scopus (139) Google Scholar). It has a pH optimum of ∼4.0 and is possibly less active than other HAases. HYAL-2 may also be exposed to the cell surface through a GPI anchor (32Lepperdinger G. Mullegger J. Kreil G. Matrix Biol. 2001; 20: 509-514Crossref PubMed Scopus (139) Google Scholar). The third HAase gene in the 3p21.3 locus is HYAL3. Although HYAL3 transcripts have been detected in brain and liver tissues, its protein product is uncharacterized (31Triggs-Raine B. Salo T.J. Zhang H. Wicklow B.A. Natowicz M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6296-6300Crossref PubMed Scopus (165) Google Scholar). Based on the sequence information deposited in GenBankTM HYAL3 is predicted to be made up of either 463 aa (accession number AF040710(gi number 2935327)) or 417 aa (accession number BC012892 (gi number 15277616)).Besides acidic HAases, PH20 (i.e. testicular HAase) is well characterized. PH20 is a sperm surface HAase that has a broad pH activity profile (pH 3.2–9.0) (33Cherr G.N. Yudin A.I. Overstreet J.W. Matrix Biol. 2001; 20: 515-525Crossref PubMed Scopus (142) Google Scholar). In addition to being a HA-degrading enzyme, PH20 may also interact with HA to increase internal calcium, possibly by binding HA through aa 205–235 (34Vines C.A., Li, M.W. Deng X. Yudin A.I. Cherr G.N. Overstreet J.W. Mol. Reprod. Dev. 2000; 60: 542-552Crossref Scopus (40) Google Scholar). This study also revealed that N-glycosylation and intrachain disulfide linkages are important for the HAase activity of PH20 (34Vines C.A., Li, M.W. Deng X. Yudin A.I. Cherr G.N. Overstreet J.W. Mol. Reprod. Dev. 2000; 60: 542-552Crossref Scopus (40) Google Scholar).Recently, the crystal structure of the bee venom HAase has been documented (35Markovic-Housley Z. Miglierini G. Soldatova L. Rizkallah P.J. Muller U. Structure. 2000; 8: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The bee HAase shares ∼30% sequence identity with human HAases. The crystal structure of bee HAase reveals a classical TIM barrel topology, where the catalytic site corresponds to Asp143 and Glu145 (aa numbering according to GenBankTM accession number AAA27730.1). Glu145is involved the cleavage of the β-1,4 glycosidic bond betweenN-acetyl-d-glucosamine andd-glucuronic acid through an acid-base catalytic mechanism. These Asp and Glu residues are conserved in all mammalian HAases (32Lepperdinger G. Mullegger J. Kreil G. Matrix Biol. 2001; 20: 509-514Crossref PubMed Scopus (139) Google Scholar). For example, in HYAL1, the putative catalytic site residues are Asp131 and Glu133, respectively, and in HYAL3, they are Asp127 and Glu129, respectively (according to GenBankTM accession numbers AAD09137.2 andAAH12892.1). Based on the amino acid homology between bee HAase and mammalian HAases and conservation of several aa residues involved in both HA binding (i.e. substrate binding groove) and its catalysis (i.e. active site), a TIM barrel topology and a similar mechanism for the catalytic cleavage of HA are likely for the mammalian HAases (32Lepperdinger G. Mullegger J. Kreil G. Matrix Biol. 2001; 20: 509-514Crossref PubMed Scopus (139) Google Scholar).In this study, we have identified several mRNA splice variants of HYAL1 and HYAL3 expressed in bladder and prostate cancer cells and in bladder and kidney tissues. These splice variants were characterized in terms of their protein product and HAase activity. We also identified a 30-aa region that is crucial for the HAase activity of HYAL1 and HYAL3 proteins.DISCUSSIONIn this study we have identified a 30-aa sequence that is well conserved in several HAases and is required for enzyme activity of at least two human HAases (i.e. HYAL1 and HYAL3). The nucleotide sequence of HYAL1 gene reveals this gene contains three exons and two introns. Exons 1 (∼1.5 kb) and 3 (0.9 kb) are relatively large compared with exon 2 (90 bp). The HYAL1 genomic sequence shows that the 90-bp sequence (nucleotides 1520–1609) that is missing in HYAL1v1 is the entire exon 2. Other HYAL1 variants appear to be generated by internal exon splicing events that usually involve exon 1. For example, the HYAL1wt described in this study is generated by an internal exon 1 splicing event, involving nucleotides 110 and 596. This splice variant has been described previously (GenBankTMaccession number AF173154). HYAL1v2 appears to be generated by internal exon splicing involving nucleotides 109 and 952 as donor/acceptor sites, respectively, both of which are present in exon 1. Generation of HYAL1v3 variant is interesting in that it involves 2 splicing events. The first is the same internal exon 1 splicing involving nucleotides 110 and 596. The second splicing involves splicing of nucleotide 1239 present in exon 1 to nucleotide 1577 that is present in exon 2. Generation of HYAL1v4 again involves an internal exon splicing event involving exon 1; both the donor (nucleotide 108) and acceptor (nucleotide 1369) sites are present in exon 1. Generation of HYAL1v5 involves internal exon 1 splicing that starts at nucleotide 109 and ends nearly at the end of exon 1 (nucleotide 1519). Thus, the analysis of HYAL1 variants demonstrates that exon 1 in HYAL1 gene has several internal donor and acceptor sites suitable for alternative mRNA splicing resulting in the generation of various HYAL1 splice variants. In addition, exon 2 can be alternatively spliced. It is interesting to note that each of these splicing events (either internal exon splicing or splicing of two exons) maintain the same open reading frame as the HYAL1 protein, resulting in different HYAL1 variant proteins.Our study on HYAL3 splice variants confirms that the 30-aa sequence that is important for enzyme activity is encoded by an independent exon. The nucleotide sequence of HYAL3 gene reveals that this gene contains four exons separated by three introns. Exon 3 is 90 bp in length and corresponds to the 90-bp v1 region (nucleotides 1090–1179). Thus, splicing out of the independent exon 3 generates HYAL3v1. As observed for HYAL1 variants, HYAL3v2 and HYAL3v3 splice variants are generated by an internal exon splicing events that involve exons 2 and 3. For example, HYAL3v2 variant is generated by an internal splicing event involving exon 2 (nucleotides 178–943 are deleted), and HYAL3v3 is generated by the same internal splicing event involving exon 2 and also splicing of the independent exon 3.It is interesting to note that different bladder and lymph node specimens and cells show differences in the pattern and types of HYAL1 and HYAL3 splice variant that are expressed. The reason for this heterogeneity is unknown at present. It is noteworthy that bladder tumors show heterogeneity in their ability to progress and recur. This heterogeneity in turn relates to the differences in the biological behavior of different bladder tumors (41Lee R. Droller M.J. Urol. Clin. North Am. 2000; 27 (, vii): 1-13Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). It is possible that the heterogeneous expression of the HYAL1 or HYAL3 variant may be related to the different biological behavior of different bladder tumors. Because in both HYAL1 and HYAL3 proteins the loss of the 30-aa sequence results in the loss of HAase activity and because the 30-aa sequence is coded by one single independent exon, the 90-bp-long independent exon in both genes seems to encode an aa sequence that is critical for HAase activity.Other studies involving site-directed mutagenesis of PH20, identification of a naturally occurring mutation in HYAL1, and the crystal structure of bee HAase have identified several aa present in different parts of HAase that are conserved and are important for activity (31Triggs-Raine B. Salo T.J. Zhang H. Wicklow B.A. Natowicz M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6296-6300Crossref PubMed Scopus (165) Google Scholar, 35Markovic-Housley Z. Miglierini G. Soldatova L. Rizkallah P.J. Muller U. Structure. 2000; 8: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 42Arming S. Strobl B. Wechselberger C. Kreil G. Eur. J. Biochem. 1997; 247: 810-814Crossref PubMed Scopus (61) Google Scholar). The crystal structure of bee HAase reveals that the insect and possibly mammalian HAases have a classical (β/α)8 TIM barrel structure (35Markovic-Housley Z. Miglierini G. Soldatova L. Rizkallah P.J. Muller U. Structure. 2000; 8: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The dominant feature of the HAase structure is a large groove that extends perpendicular to the barrel axis. In bee HAase, loops following the β strands 2, 3, and 4 form one wall of the groove, and those of 1, 5, 6, and 7 form the other wall. This groove is large enough to accommodate a hexasaccharide (35Markovic-Housley Z. Miglierini G. Soldatova L. Rizkallah P.J. Muller U. Structure. 2000; 8: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Co-crystallization of bee HAase with a hexasaccharide shows that the catalytic site that cleaves the glycosidic bond betweenN-acetyl-d-glucosamine andd-glucuronic acid lies in aa residues Asp143and Glu145 (numbering according to GenBankTMaccession number AAA27730.1). In a substrate-assisted acid-base catalytic mechanism, Glu145 acts as the proton donor, and the N-acetyl group of the substrate acts as the nucleophile. In all human HAases, this Glu residue is conserved along with Asp and is believed to be responsible for substrate cleavage (32Lepperdinger G. Mullegger J. Kreil G. Matrix Biol. 2001; 20: 509-514Crossref PubMed Scopus (139) Google Scholar). In the HYAL1 sequence this Glu is aa 131 (numbering according to GenBankTM accession number AAD09137.2), and in HYAL3 it is aa 129 (numbering according to GenBankTM accession numberAAH12892.1). Thus, the 30-aa sequence that we have identified in HYAL1 (aa 301–300) and in HYAL3 (aa 299–328) most likely is not a part of the catalytic site.Based on the bee HAase crystal structure, the 30-aa sequence from aa 313 to 342 (Fig. 11), that is homologous to the 30-aa sequences in HYAL1 and HYAL3, forms β sheets 6 and 7, α-helix 8, and the loops in between (35Markovic-Housley Z. Miglierini G. Soldatova L. Rizkallah P.J. Muller U. Structure. 2000; 8: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Because the loops following β strands 6 and 7 are involved in forming one of the walls of the substrate binding groove, loss of the 30-aa sequence in HYAL1 and HYAL3 proteins may result in the loss of substrate binding. The substrate-associated catalytic cleavage of the glycosidic bond betweenN-acetyl-d-glucosamine and d-glucuronic acid requires accurate positioning of theN-acetyl side chain of the substrate with respect to the catalytic site (35Markovic-Housley Z. Miglierini G. Soldatova L. Rizkallah P.J. Muller U. Structure. 2000; 8: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). This is achieved by two hydrogen bonding interactions and a hydrophobic interaction. The crystal structure shows that in bee HAase, Trp333 is involved in hydrophobic interaction with the N-acetyl side chain (35Markovic-Housley Z. Miglierini G. Soldatova L. Rizkallah P.J. Muller U. Structure. 2000; 8: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). This Trp333 corresponds to Trp321 in HYAL1 and Trp319 in HYAL3, both of which are present in the respective 30-aa sequences (Fig. 11). This Trp residue is also conserved in all HAases and other chitinolytic enzymes (43Tews I. Perrakis A. Oppenheim A. Dauter Z. Wilson K.S. Vorgias C.E. Nat. Struct. Biol. 1996; 3: 638-648Crossref PubMed Scopus (324) Google Scholar, 44Tews I. Terwisscha van Scheltinga A.C. Perrakis A. Wilson K.S. Dijkstra B.W. J. Am. Chem. Soc. 1997; 119: 7954-7959Crossref Scopus (270) Google Scholar). Thus, the absence of this Trp residue in HYAL1v1 and HYAL3v1 mutant proteins may lead to improper positioning of the substrate resulting in no catalysis. It remains to be determined why this 30-aa sequence is so well conserved in various HAases and what role other conserved residues play in terms of substrate binding and catalysis. It should be noted that the loss of β sheets 6 and 7, α-helix 8, and the loops in between, because of the 30-aa deletion in HYAL1v1 and HYAL3v1, may also result in complete loss of the TIM barrel structure, making these proteins enzymatically inactive.Other HYAL1 and HYAL3 variant proteins also illustrate the importance of various structural domains for HAase activity. For example, HYAL1v3 variant, which contains the putative catalytic site (i.e.Asp129 and Glu131) but lacks aa 208–435, has no HAase activity. It is likely that the HYAL1v3 protein does not form a proper TIM barrel structure, and at the very least, it lacks the substrate-binding groove. HYAL1v2 (aa 183–435) and HYAL1v4 (aa 260–435) proteins retain the 30-aa sequence and also Glu268, which has been shown to be critical for HAase activity. However, these variants are enzymatically inactive, because they lack the putative catalytic site as well as parts of the substrate-binding groove. A similar situation may hold true for HYAL3v2 variant (aa 251–435), which also does not have any HAase activity. The variants HYAL1v5 (aa 340–435) and HYAL3v3 (aa 251–435 but lacking aa 299–328) lack more than two-thirds of the respective molecules and hence will not fold properly, lack the HAase catalytic site, and have no substrate binding groove. Thus, this study illustrates the involvement of several structural domains that are conserved in various HAases and are important for enzyme activity.Detection of various HYAL1/HYAL3 variants in bladder and prostate cancer lines, bladder tumor tissues, as well as normal kidney tissue suggest that the expression of functionally active HAase in various cells and tissues may be regulated by alternative mRNA splicing. We have previously shown that HAase levels are elevated (3–7-fold) in the urine of bladder cancer patients who have high grade (i.e.G2/G3) disease (39Pham H.T. Block N.L. Lokeshwar V.B. Cancer Res. 1997; 57 (; Correction (1997) Cancer Res.57, 1662): 778-783PubMed Google Scholar). Furthermore, in a study of 504 patients, we demonstrated that the HAase level serves as a highly sensitive (82%) and specific (83%) marker for detecting G2/G3 bladder cancer (18Lokeshwar V.B. Obek C. Pham H.T. Wei D. Young M.J. Duncan R.C. Soloway M.S. Block N.L. J. Urol. 2000; 163: 348-356Crossref PubMed Scopus (173) Google Scholar). Urinary HAase levels together with HA levels (i.e. HA-HAase test) are sensitive and specific in detecting bladder cancer and monitoring its recurrence (18Lokeshwar V.B. Obek C. Pham H.T. Wei D. Young M.J. Duncan R.C. Soloway M.S. Block N.L. J. Urol. 2000; 163: 348-356Crossref PubMed Scopus (173) Google Scholar, 45Lokeshwar V.B. Schroeder G.L. Selzer M.G. Hautmann S.H. Posey J.T. Duncan R.C. Watson R. Rose L. Markowitz S. Soloway M.S. Cancer. 2002; 95: 61-72Crossref PubMed Scopus (97) Google Scholar). We have also shown that both invasive bladder and prostate cancer cells secrete high levels of HAase activity, and HYAL1 is the major HAase expressed in these carcinomas (17Lokeshwar V.B. Rubinowicz D. Schroeder G.L. Forgacs E. Minna J.D. Block N.L. Nadji M. Lokeshwar B.L. J. Biol. Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 30Lokeshwar V.B. Young M.J. Gouddarzi G. Iida N. Yudin A.I. Cherr G.N. Selzer M.G. Cancer Res. 1999; 59: 4464-4470PubMed Google Scholar, 46Lokeshwar V.B. Soloway M.S. Block N.L. Cancer Lett. 1998; 131: 21-27Crossref PubMed Scopus (35) Google Scholar). Consistent with these observations, in this study we observed that the full-length HYAL1wt transcript that encodes a functional HAase is expressed only in bladder and prostate cancer cells, G2/G3 bladder tumor tissues, and lymph node specimens showing tumor invasion. In normal bladder and G1 bladder tumor tissues, in a normal bladder primary culture, and in bladder tissues showing no evidence of tumor, the major HYAL1 transcript that is expressed is the HYAL1v5 variant. Because this transcript or other HYAL1 variant transcripts (i.e. HYAL1v2-v4) that are also expressed in some of these specimens do not encode a functional HAase, it may explain why in these tissues or cells no HAase activity and wild type HYAL1 protein are detected (30Lokeshwar V.B. Young M.J. Gouddarzi G. Iida N. Yudin A.I. Cherr G.N. Selzer M.G. Cancer Res. 1999; 59: 4464-4470PubMed Google Scholar, 46Lokeshwar V.B. Soloway M.S. Block N.L. Cancer Lett. 1998; 131: 21-27Crossref PubMed Scopus (35) Google Scholar, 47Hautmann S.H. Lokeshwar V.B. Schroeder G.L. Civantos F. Duncan R.C. Gnann R. Friedrich M.G. Soloway M.S. J. Urol. 2001; 165: 2068-2074Crossref PubMed Google Scholar).HAase protein has been shown to be associated with tumor angiogenesis and/or invasion (48Liu D. Pearlman E. Diacnou E. Guo K. Mori H. Haqqi T. Markowitz S. Wilson J. Sy M.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7832-7837Crossref PubMed Scopus (200) Google Scholar). However, because HYAL1 (and also HYAL2 and HYAL3) is present on chromosome 3p 21.3 locus, and this region is a critical tumor homozygous deletion region in lung and breast cancers, it has been suggested that HYAL1 may be a tumor suppressor (7Csoka A.B. Frost G.I. Stern R. Matrix Biol. 2001; 20: 499-508Crossref PubMed Scopus (474) Google Scholar, 49Frost G.I. Mohapatra G. Wong T.M. Csoka A.B. Gray J.W. Stern R. Oncogene. 2000; 19: 870-877Crossref PubMed Scopus (75) Google Scholar). A recent study clearly demonstrates that the RASSF1 gene and not HYAL1 is the tumor suppressor gene present in that locus (50Ji L. Nishizaki M. Gao B. Burbee D. Kondo M. Kamibayashi C., Xu, K. Yen N. Atkinson E.N. Fang B. Lerman M.I. Roth J.A. Minna J.D. Cancer Res. 2002; 262: 2715-2720Google Scholar). Thus, several observations, including elevation of HAase levels in bladder and prostate tumors (18Lokeshwar V.B. Obek C. Pham H.T. Wei D. Young M.J. Duncan R.C. Soloway M.S. Block N.L. J. Urol. 2000; 163: 348-356Crossref PubMed Scopus (173)

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