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• W2045014705 abstract "Using Expressed Sequence Tags (ESTs) deposited in the data banks, a cDNA has been assembled that encodes a protein related to the hyaluronidases from bee venom and mammalian sperm. Expression of this cDNA yielded a polypeptide termed HYAL2, which is located in lysosomes. The HYAL2 protein was shown to have hyaluronidase activity below pH 4. However, it only hydrolyzed hyaluronan of high molecular mass from umbilical cord, rooster comb, and a Streptococcus strain. The reaction product was a polysaccharide of about 20 kDa, which was further hydrolyzed to small oligosaccharides by the sperm hyaluronidase. Conversely, hyaluronan fragments from vitreous humor, which had a molecular mass of about 20 kDa, were not cleaved by the HYAL2 enzyme to any detectable extent. These results provide evidence for the existence of structural domains in hyaluronan, which are resistant to the action of this enzyme. The structural and functional implications of these findings are discussed. Using Expressed Sequence Tags (ESTs) deposited in the data banks, a cDNA has been assembled that encodes a protein related to the hyaluronidases from bee venom and mammalian sperm. Expression of this cDNA yielded a polypeptide termed HYAL2, which is located in lysosomes. The HYAL2 protein was shown to have hyaluronidase activity below pH 4. However, it only hydrolyzed hyaluronan of high molecular mass from umbilical cord, rooster comb, and a Streptococcus strain. The reaction product was a polysaccharide of about 20 kDa, which was further hydrolyzed to small oligosaccharides by the sperm hyaluronidase. Conversely, hyaluronan fragments from vitreous humor, which had a molecular mass of about 20 kDa, were not cleaved by the HYAL2 enzyme to any detectable extent. These results provide evidence for the existence of structural domains in hyaluronan, which are resistant to the action of this enzyme. The structural and functional implications of these findings are discussed. Hyaluronidases have been isolated from many different sources such as mammalian testes and serum, snake and insect venoms, salivary glands of leeches, and pathogenic streptococci (1Frost G.I. Csóka T.B. Stern R. Trends Glycosci. Glycotech. 1997; 8: 419-434Crossref Scopus (101) Google Scholar, 2Kreil G. Protein Sci. 1995; 4: 1666-1669Crossref PubMed Scopus (306) Google Scholar, 3Meyer K. Boyer P.D. Hyaluronidases. V. Academic Press, New York1971Google Scholar). These enzymes degrade hyaluronan (hyaluronic acid (HA) 1The abbreviations used are: HAhyaluronan (hyaluronic acid)PCRpolymerase chain reactionESTexpressed sequence tagvvvaccinia virusEGFPgreen fluorescent protein.1The abbreviations used are: HAhyaluronan (hyaluronic acid)PCRpolymerase chain reactionESTexpressed sequence tagvvvaccinia virusEGFPgreen fluorescent protein.), a glycosaminoglycan present in the extracellular matrix of vertebrates to oligosaccharides (4Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2049) Google Scholar). The sequences of two related hyaluronidases from animal cells have recently be elucidated via cDNA cloning. These are the enzymes from honeybee venom (5Gmachl M. Kreil G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3569-3573Crossref PubMed Scopus (177) Google Scholar) and from mammalian testis (6Gmachl M. Sagan S. Ketter S. Kreil G. FEBS Lett. 1993; 336: 545-548Crossref PubMed Scopus (122) Google Scholar, 7Lin Y. Mahan K. Lathrop W.F. Myles D.G. Primakoff P. J. Cell Biol. 1994; 125: 1157-1163Crossref PubMed Scopus (219) Google Scholar). The testicular enzyme originally termed PH-20 (8Lathrop W.F. Carmichael E.P. Myles D.G. Primakoff P. J. Cell Biol. 1990; 111: 2939-2949Crossref PubMed Scopus (117) Google Scholar) is located at the head of the sperm; upon contact with the egg, it hydrolyzes the HA present in its outermost cumulus layer (9Cherr G.N. Meyers S.A. Yudin A.I. VandeVoort C.A. Myles D.G. Primakoff P. Overstreet J.W. Dev. Biol. 1996; 175: 142-153Crossref PubMed Scopus (99) Google Scholar). The PH-20 protein is normally expressed only in mammalian testis. However, it has recently been shown that this enzyme is present in some tumor cells (10Liu D. Pearlman E. Diaconu 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). A related hyaluronidase termed HYAL1, which is present in human serum, has recently been characterized (11Frost G.I. Csóka T.B. Wong T. Stern R. Biochem. Biophys. Res. Commun. 1997; 236: 10-15Crossref PubMed Scopus (208) Google Scholar).In recent years, several partial cDNA sequences have been deposited in the data banks that are derived from genes that potentially code for polypeptides related to the PH-20 hyaluronidase. Three of these genes are located close to each other on chromosome region 3p21.3 (12Latif F. Duh F.M. Bader S. Sekido Y. Li H. Geil L. Zbar B. Minna J.D. Lerman M.I. Hum. Genet. 1997; 99: 334-341Crossref PubMed Scopus (24) Google Scholar). This region is deleted in some cell lines from small cell carcinomas of the lung (13Daly M.C. Xiang R.H. Buchhagen D. Hensel C.H. Garcia D.K. Killary A.M. Minna J.D. Naylor S.L. Oncogene. 1993; 8: 1721-1729PubMed Google Scholar). The genes were thus provisionally termed LuCa-1, -2, and -3. The serum enzyme HYAL1 is the product of the LuCa-1 gene (11Frost G.I. Csóka T.B. Wong T. Stern R. Biochem. Biophys. Res. Commun. 1997; 236: 10-15Crossref PubMed Scopus (208) Google Scholar). Starting from commercially available ESTs we have now assembled the complete LuCa-2cDNA. Expression of this cDNA yields a hyaluronidase with a rather unusual substrate specificity, which is located in lysosomes. It is proposed to replace the term LuCa-2 by HYAL2for both the gene and its product.DISCUSSIONThe human HYAL2 cDNA encodes a mature polypeptide of 452 amino acids that shows 36.5% identity with the PH-20 hyaluronidase present on the head of spermatozoa. At the nucleotide level, the identity is 43.1%. The HYAL2 gene is located on chromosome 3p21.3 in a region that is deleted in most small cell carcinomas of the lung as well as other tumor cells (12Latif F. Duh F.M. Bader S. Sekido Y. Li H. Geil L. Zbar B. Minna J.D. Lerman M.I. Hum. Genet. 1997; 99: 334-341Crossref PubMed Scopus (24) Google Scholar, 13Daly M.C. Xiang R.H. Buchhagen D. Hensel C.H. Garcia D.K. Killary A.M. Minna J.D. Naylor S.L. Oncogene. 1993; 8: 1721-1729PubMed Google Scholar). This gene was tentatively termedLuCa-2; in view of the results presented in this communication, we propose to now call it HYAL2. The hyaluronidase present in mouse (24Sampson P.M. Rochester C.L. Freundlich B. Elias J.A. J. Clin. Invest. 1992; 90: 1492-1503Crossref PubMed Scopus (134) Google Scholar) and human (11Frost G.I. Csóka T.B. Wong T. Stern R. Biochem. Biophys. Res. Commun. 1997; 236: 10-15Crossref PubMed Scopus (208) Google Scholar) serum was termed HYAL1 earlier. This enzyme is apparently secreted by a variety of cells.The human HYAL2 gene is expressed in all cells tested, the sole exception being adult brain. It is noteworthy that several of the ESTs encoding part of the HYAL2 enzyme have been isolated from infant human brain. The expression of the HYAL2 gene may thus be developmentally regulated. Indeed, the mouse HYAL2 mRNA is present in brains from embryos but disappears for unknown reasons soon after birth. 2G. Lepperdinger, B. Strobl, and G. Kreil, unpublished experiments.Using recombinant vv, the HYAL2 protein has been expressed in several cell lines; here we present the results obtained with HeLa and C6 glioma cell. The HYAL2 cDNA codes for a preprotein with an amino-terminal signal peptide, yet the protein is not secreted into the media to a measurable extent. The product of this gene is a soluble hyaluronidase that resembles the lysosomal enzyme present in HeLa cells. Both have a pH optimum below 4 and react with the same antiserum. A fusion protein of HYAL2 and EGFP could be shown to be localized in lysosomes of C6 glioma cells. We conclude from these results that HYAL2 encodes a lysosomal hyaluronidase present in many cell types.It was apparent from initial turbidity measurements that the activity of HYAL2 was rather low compared with that of the testicular PH-20 enzyme. Subsequent experiments with a gel electrophoresis assay yielded a totally unexpected result. The HYAL2 enzyme hydrolyzed only HA of high molecular mass, as is present in umbilical cord, rooster comb, and the coat of a Streptococcus strain. The reaction product was a polysaccharide of about 20 kDa, which corresponds to 50–60 disaccharide units. After heating and rapid cooling, no further hydrolysis by freshly added HYAL2 enzyme could be observed. However, upon subsequent addition of the PH-20 enzyme, this intermediate was quantitatively degraded to small oligosaccharides. HA of smaller size as well as a synthetic product made in vitro with the DG42 HA synthase were not degraded to a detectable extent by the HYAL2 enzyme. A similar finding has been reported by Sampson et al. (24Sampson P.M. Rochester C.L. Freundlich B. Elias J.A. J. Clin. Invest. 1992; 90: 1492-1503Crossref PubMed Scopus (134) Google Scholar), who showed that human fibroblasts contain a hyaluronidase that degrades high molecular weight HA to products with a size of 10–40 kDa. Interestingly, in these experiments, binding of HA to the cells appeared to be rate-limiting for degradation.The results obtained with the HYAL2 enzyme are of some interest from both a structural and a functional point of view. First, it appears that ordered domains are present in HA, which are resistant to the action of the HYAL2 enzyme. HA is a linear polymer that behaves in solution as a random coil containing large amounts of solvent (25Laurent T.C. CIBA Found. Symp. 1989; 143: 1-5PubMed Google Scholar). However, the flexibility of this coil is locally reduced by the presence of hydrogen bonds (26Scott J.E. CIBA Found. Symp. 1989; 143: 6-15PubMed Google Scholar). Moreover, several groups have presented evidence that HA chains can form a complex network containing numerous helical structures (27Arnott S. Mitra A.K. J. Mol. Biol. 1983; 169: 861-872Crossref PubMed Scopus (58) Google Scholar, 28Scott J.E. Cummings C. Greiling H. Stuhlsatz H.W. Gregory J.D. Damle S.P. Int. J. Biol. Macromol. 1990; 12: 180-184Crossref PubMed Scopus (30) Google Scholar). Antiparallel helices have indeed been observed in x-ray diffraction studies with HA films (29Mitra A.K. Arnott S. Sheehan J.K. J. Mol. Biol. 1983; 169: 813-827Crossref PubMed Scopus (31) Google Scholar). Evidence from physico-chemical studies suggests that similar structures exist in HA solutions (25Laurent T.C. CIBA Found. Symp. 1989; 143: 1-5PubMed Google Scholar, 30Scott J.E. Cummings C. Brass A. Chen Y. Biochem. J. 1991; 274: 699-705Crossref PubMed Scopus (252) Google Scholar). Our results on the activity of the HYAL2 enzyme provide the first biochemical data in support of defined domains in HA.Second, HA fragments generated by the HYAL2 enzyme may have distinct biological functions. One could be the stimulation of angiogenesis, which has been observed in different experimental situations (10Liu D. Pearlman E. Diaconu 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, 31West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (961) Google Scholar). Moreover, it is of some interest that HA fragments can induce the expression of enzymes such as nitric oxide synthase via a NF-κB/I-κBα autoregulatory loop in murine macrophages (32Noble P.W. McKee C.M. Cowman M. Shin H.S. J. Exp. Med. 1996; 183: 2373-2378Crossref PubMed Scopus (277) Google Scholar, 33McKee C.M. Lowenstein C.J. Horton M.R. Wu J. Bao C. Clin B.Y. Choi A.M.K. Noble P.W. J. Biol. Chem. 1997; 272: 8013-8018Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The participation of HYAL2 in this signaling process can now readily be tested. Hyaluronidases have been isolated from many different sources such as mammalian testes and serum, snake and insect venoms, salivary glands of leeches, and pathogenic streptococci (1Frost G.I. Csóka T.B. Stern R. Trends Glycosci. Glycotech. 1997; 8: 419-434Crossref Scopus (101) Google Scholar, 2Kreil G. Protein Sci. 1995; 4: 1666-1669Crossref PubMed Scopus (306) Google Scholar, 3Meyer K. Boyer P.D. Hyaluronidases. V. Academic Press, New York1971Google Scholar). These enzymes degrade hyaluronan (hyaluronic acid (HA) 1The abbreviations used are: HAhyaluronan (hyaluronic acid)PCRpolymerase chain reactionESTexpressed sequence tagvvvaccinia virusEGFPgreen fluorescent protein.1The abbreviations used are: HAhyaluronan (hyaluronic acid)PCRpolymerase chain reactionESTexpressed sequence tagvvvaccinia virusEGFPgreen fluorescent protein.), a glycosaminoglycan present in the extracellular matrix of vertebrates to oligosaccharides (4Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2049) Google Scholar). The sequences of two related hyaluronidases from animal cells have recently be elucidated via cDNA cloning. These are the enzymes from honeybee venom (5Gmachl M. Kreil G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3569-3573Crossref PubMed Scopus (177) Google Scholar) and from mammalian testis (6Gmachl M. Sagan S. Ketter S. Kreil G. FEBS Lett. 1993; 336: 545-548Crossref PubMed Scopus (122) Google Scholar, 7Lin Y. Mahan K. Lathrop W.F. Myles D.G. Primakoff P. J. Cell Biol. 1994; 125: 1157-1163Crossref PubMed Scopus (219) Google Scholar). The testicular enzyme originally termed PH-20 (8Lathrop W.F. Carmichael E.P. Myles D.G. Primakoff P. J. Cell Biol. 1990; 111: 2939-2949Crossref PubMed Scopus (117) Google Scholar) is located at the head of the sperm; upon contact with the egg, it hydrolyzes the HA present in its outermost cumulus layer (9Cherr G.N. Meyers S.A. Yudin A.I. VandeVoort C.A. Myles D.G. Primakoff P. Overstreet J.W. Dev. Biol. 1996; 175: 142-153Crossref PubMed Scopus (99) Google Scholar). The PH-20 protein is normally expressed only in mammalian testis. However, it has recently been shown that this enzyme is present in some tumor cells (10Liu D. Pearlman E. Diaconu 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). A related hyaluronidase termed HYAL1, which is present in human serum, has recently been characterized (11Frost G.I. Csóka T.B. Wong T. Stern R. Biochem. Biophys. Res. Commun. 1997; 236: 10-15Crossref PubMed Scopus (208) Google Scholar). hyaluronan (hyaluronic acid) polymerase chain reaction expressed sequence tag vaccinia virus green fluorescent protein. hyaluronan (hyaluronic acid) polymerase chain reaction expressed sequence tag vaccinia virus green fluorescent protein. In recent years, several partial cDNA sequences have been deposited in the data banks that are derived from genes that potentially code for polypeptides related to the PH-20 hyaluronidase. Three of these genes are located close to each other on chromosome region 3p21.3 (12Latif F. Duh F.M. Bader S. Sekido Y. Li H. Geil L. Zbar B. Minna J.D. Lerman M.I. Hum. Genet. 1997; 99: 334-341Crossref PubMed Scopus (24) Google Scholar). This region is deleted in some cell lines from small cell carcinomas of the lung (13Daly M.C. Xiang R.H. Buchhagen D. Hensel C.H. Garcia D.K. Killary A.M. Minna J.D. Naylor S.L. Oncogene. 1993; 8: 1721-1729PubMed Google Scholar). The genes were thus provisionally termed LuCa-1, -2, and -3. The serum enzyme HYAL1 is the product of the LuCa-1 gene (11Frost G.I. Csóka T.B. Wong T. Stern R. Biochem. Biophys. Res. Commun. 1997; 236: 10-15Crossref PubMed Scopus (208) Google Scholar). Starting from commercially available ESTs we have now assembled the complete LuCa-2cDNA. Expression of this cDNA yields a hyaluronidase with a rather unusual substrate specificity, which is located in lysosomes. It is proposed to replace the term LuCa-2 by HYAL2for both the gene and its product. DISCUSSIONThe human HYAL2 cDNA encodes a mature polypeptide of 452 amino acids that shows 36.5% identity with the PH-20 hyaluronidase present on the head of spermatozoa. At the nucleotide level, the identity is 43.1%. The HYAL2 gene is located on chromosome 3p21.3 in a region that is deleted in most small cell carcinomas of the lung as well as other tumor cells (12Latif F. Duh F.M. Bader S. Sekido Y. Li H. Geil L. Zbar B. Minna J.D. Lerman M.I. Hum. Genet. 1997; 99: 334-341Crossref PubMed Scopus (24) Google Scholar, 13Daly M.C. Xiang R.H. Buchhagen D. Hensel C.H. Garcia D.K. Killary A.M. Minna J.D. Naylor S.L. Oncogene. 1993; 8: 1721-1729PubMed Google Scholar). This gene was tentatively termedLuCa-2; in view of the results presented in this communication, we propose to now call it HYAL2. The hyaluronidase present in mouse (24Sampson P.M. Rochester C.L. Freundlich B. Elias J.A. J. Clin. Invest. 1992; 90: 1492-1503Crossref PubMed Scopus (134) Google Scholar) and human (11Frost G.I. Csóka T.B. Wong T. Stern R. Biochem. Biophys. Res. Commun. 1997; 236: 10-15Crossref PubMed Scopus (208) Google Scholar) serum was termed HYAL1 earlier. This enzyme is apparently secreted by a variety of cells.The human HYAL2 gene is expressed in all cells tested, the sole exception being adult brain. It is noteworthy that several of the ESTs encoding part of the HYAL2 enzyme have been isolated from infant human brain. The expression of the HYAL2 gene may thus be developmentally regulated. Indeed, the mouse HYAL2 mRNA is present in brains from embryos but disappears for unknown reasons soon after birth. 2G. Lepperdinger, B. Strobl, and G. Kreil, unpublished experiments.Using recombinant vv, the HYAL2 protein has been expressed in several cell lines; here we present the results obtained with HeLa and C6 glioma cell. The HYAL2 cDNA codes for a preprotein with an amino-terminal signal peptide, yet the protein is not secreted into the media to a measurable extent. The product of this gene is a soluble hyaluronidase that resembles the lysosomal enzyme present in HeLa cells. Both have a pH optimum below 4 and react with the same antiserum. A fusion protein of HYAL2 and EGFP could be shown to be localized in lysosomes of C6 glioma cells. We conclude from these results that HYAL2 encodes a lysosomal hyaluronidase present in many cell types.It was apparent from initial turbidity measurements that the activity of HYAL2 was rather low compared with that of the testicular PH-20 enzyme. Subsequent experiments with a gel electrophoresis assay yielded a totally unexpected result. The HYAL2 enzyme hydrolyzed only HA of high molecular mass, as is present in umbilical cord, rooster comb, and the coat of a Streptococcus strain. The reaction product was a polysaccharide of about 20 kDa, which corresponds to 50–60 disaccharide units. After heating and rapid cooling, no further hydrolysis by freshly added HYAL2 enzyme could be observed. However, upon subsequent addition of the PH-20 enzyme, this intermediate was quantitatively degraded to small oligosaccharides. HA of smaller size as well as a synthetic product made in vitro with the DG42 HA synthase were not degraded to a detectable extent by the HYAL2 enzyme. A similar finding has been reported by Sampson et al. (24Sampson P.M. Rochester C.L. Freundlich B. Elias J.A. J. Clin. Invest. 1992; 90: 1492-1503Crossref PubMed Scopus (134) Google Scholar), who showed that human fibroblasts contain a hyaluronidase that degrades high molecular weight HA to products with a size of 10–40 kDa. Interestingly, in these experiments, binding of HA to the cells appeared to be rate-limiting for degradation.The results obtained with the HYAL2 enzyme are of some interest from both a structural and a functional point of view. First, it appears that ordered domains are present in HA, which are resistant to the action of the HYAL2 enzyme. HA is a linear polymer that behaves in solution as a random coil containing large amounts of solvent (25Laurent T.C. CIBA Found. Symp. 1989; 143: 1-5PubMed Google Scholar). However, the flexibility of this coil is locally reduced by the presence of hydrogen bonds (26Scott J.E. CIBA Found. Symp. 1989; 143: 6-15PubMed Google Scholar). Moreover, several groups have presented evidence that HA chains can form a complex network containing numerous helical structures (27Arnott S. Mitra A.K. J. Mol. Biol. 1983; 169: 861-872Crossref PubMed Scopus (58) Google Scholar, 28Scott J.E. Cummings C. Greiling H. Stuhlsatz H.W. Gregory J.D. Damle S.P. Int. J. Biol. Macromol. 1990; 12: 180-184Crossref PubMed Scopus (30) Google Scholar). Antiparallel helices have indeed been observed in x-ray diffraction studies with HA films (29Mitra A.K. Arnott S. Sheehan J.K. J. Mol. Biol. 1983; 169: 813-827Crossref PubMed Scopus (31) Google Scholar). Evidence from physico-chemical studies suggests that similar structures exist in HA solutions (25Laurent T.C. CIBA Found. Symp. 1989; 143: 1-5PubMed Google Scholar, 30Scott J.E. Cummings C. Brass A. Chen Y. Biochem. J. 1991; 274: 699-705Crossref PubMed Scopus (252) Google Scholar). Our results on the activity of the HYAL2 enzyme provide the first biochemical data in support of defined domains in HA.Second, HA fragments generated by the HYAL2 enzyme may have distinct biological functions. One could be the stimulation of angiogenesis, which has been observed in different experimental situations (10Liu D. Pearlman E. Diaconu 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, 31West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (961) Google Scholar). Moreover, it is of some interest that HA fragments can induce the expression of enzymes such as nitric oxide synthase via a NF-κB/I-κBα autoregulatory loop in murine macrophages (32Noble P.W. McKee C.M. Cowman M. Shin H.S. J. Exp. Med. 1996; 183: 2373-2378Crossref PubMed Scopus (277) Google Scholar, 33McKee C.M. Lowenstein C.J. Horton M.R. Wu J. Bao C. Clin B.Y. Choi A.M.K. Noble P.W. J. Biol. Chem. 1997; 272: 8013-8018Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The participation of HYAL2 in this signaling process can now readily be tested. The human HYAL2 cDNA encodes a mature polypeptide of 452 amino acids that shows 36.5% identity with the PH-20 hyaluronidase present on the head of spermatozoa. At the nucleotide level, the identity is 43.1%. The HYAL2 gene is located on chromosome 3p21.3 in a region that is deleted in most small cell carcinomas of the lung as well as other tumor cells (12Latif F. Duh F.M. Bader S. Sekido Y. Li H. Geil L. Zbar B. Minna J.D. Lerman M.I. Hum. Genet. 1997; 99: 334-341Crossref PubMed Scopus (24) Google Scholar, 13Daly M.C. Xiang R.H. Buchhagen D. Hensel C.H. Garcia D.K. Killary A.M. Minna J.D. Naylor S.L. Oncogene. 1993; 8: 1721-1729PubMed Google Scholar). This gene was tentatively termedLuCa-2; in view of the results presented in this communication, we propose to now call it HYAL2. The hyaluronidase present in mouse (24Sampson P.M. Rochester C.L. Freundlich B. Elias J.A. J. Clin. Invest. 1992; 90: 1492-1503Crossref PubMed Scopus (134) Google Scholar) and human (11Frost G.I. Csóka T.B. Wong T. Stern R. Biochem. Biophys. Res. Commun. 1997; 236: 10-15Crossref PubMed Scopus (208) Google Scholar) serum was termed HYAL1 earlier. This enzyme is apparently secreted by a variety of cells. The human HYAL2 gene is expressed in all cells tested, the sole exception being adult brain. It is noteworthy that several of the ESTs encoding part of the HYAL2 enzyme have been isolated from infant human brain. The expression of the HYAL2 gene may thus be developmentally regulated. Indeed, the mouse HYAL2 mRNA is present in brains from embryos but disappears for unknown reasons soon after birth. 2G. Lepperdinger, B. Strobl, and G. Kreil, unpublished experiments. Using recombinant vv, the HYAL2 protein has been expressed in several cell lines; here we present the results obtained with HeLa and C6 glioma cell. The HYAL2 cDNA codes for a preprotein with an amino-terminal signal peptide, yet the protein is not secreted into the media to a measurable extent. The product of this gene is a soluble hyaluronidase that resembles the lysosomal enzyme present in HeLa cells. Both have a pH optimum below 4 and react with the same antiserum. A fusion protein of HYAL2 and EGFP could be shown to be localized in lysosomes of C6 glioma cells. We conclude from these results that HYAL2 encodes a lysosomal hyaluronidase present in many cell types. It was apparent from initial turbidity measurements that the activity of HYAL2 was rather low compared with that of the testicular PH-20 enzyme. Subsequent experiments with a gel electrophoresis assay yielded a totally unexpected result. The HYAL2 enzyme hydrolyzed only HA of high molecular mass, as is present in umbilical cord, rooster comb, and the coat of a Streptococcus strain. The reaction product was a polysaccharide of about 20 kDa, which corresponds to 50–60 disaccharide units. After heating and rapid cooling, no further hydrolysis by freshly added HYAL2 enzyme could be observed. However, upon subsequent addition of the PH-20 enzyme, this intermediate was quantitatively degraded to small oligosaccharides. HA of smaller size as well as a synthetic product made in vitro with the DG42 HA synthase were not degraded to a detectable extent by the HYAL2 enzyme. A similar finding has been reported by Sampson et al. (24Sampson P.M. Rochester C.L. Freundlich B. Elias J.A. J. Clin. Invest. 1992; 90: 1492-1503Crossref PubMed Scopus (134) Google Scholar), who showed that human fibroblasts contain a hyaluronidase that degrades high molecular weight HA to products with a size of 10–40 kDa. Interestingly, in these experiments, binding of HA to the cells appeared to be rate-limiting for degradation. The results obtained with the HYAL2 enzyme are of some interest from both a structural and a functional point of view. First, it appears that ordered domains are present in HA, which are resistant to the action of the HYAL2 enzyme. HA is a linear polymer that behaves in solution as a random coil containing large amounts of solvent (25Laurent T.C. CIBA Found. Symp. 1989; 143: 1-5PubMed Google Scholar). However, the flexibility of this coil is locally reduced by the presence of hydrogen bonds (26Scott J.E. CIBA Found. Symp. 1989; 143: 6-15PubMed Google Scholar). Moreover, several groups have presented evidence that HA chains can form a complex network containing numerous helical structures (27Arnott S. Mitra A.K. J. Mol. Biol. 1983; 169: 861-872Crossref PubMed Scopus (58) Google Scholar, 28Scott J.E. Cummings C. Greiling H. Stuhlsatz H.W. Gregory J.D. Damle S.P. Int. J. Biol. Macromol. 1990; 12: 180-184Crossref PubMed Scopus (30) Google Scholar). Antiparallel helices have indeed been observed in x-ray diffraction studies with HA films (29Mitra A.K. Arnott S. Sheehan J.K. J. Mol. Biol. 1983; 169: 813-827Crossref PubMed Scopus (31) Google Scholar). Evidence from physico-chemical studies suggests that similar structures exist in HA solutions (25Laurent T.C. CIBA Found. Symp. 1989; 143: 1-5PubMed Google Scholar, 30Scott J.E. Cummings C. Brass A. Chen Y. Biochem. J. 1991; 274: 699-705Crossref PubMed Scopus (252) Google Scholar). Our results on the activity of the HYAL2 enzyme provide the first biochemical data in support of defined domains in HA. Second, HA fragments generated by the HYAL2 enzyme may have distinct biological functions. One could be the stimulation of angiogenesis, which has been observed in different experimental situations (10Liu D. Pearlman E. Diaconu 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, 31West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (961) Google Scholar). Moreover, it is of some interest that HA fragments can induce the expression of enzymes such as nitric oxide synthase via a NF-κB/I-κBα autoregulatory loop in murine macrophages (32Noble P.W. McKee C.M. Cowman M. Shin H.S. J. Exp. Med. 1996; 183: 2373-2378Crossref PubMed Scopus (277) Google Scholar, 33McKee C.M. Lowenstein C.J. Horton M.R. Wu J. Bao C. Clin B.Y. Choi A.M.K. Noble P.W. J. Biol. Chem. 1997; 272: 8013-8018Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The participation of HYAL2 in this signaling process can now readily be tested. We thank Dr. Thomas Weiger (University of Salzburg) for help and assistance with the confocal microscope. HA preparations of different molecular mass were kindly supplied by Dr. P. Heldin (University of Uppsala) and Dr. O. Wik (Amersham Pharmacia Biotech and The Upjohn Co.)." @default.
• W2045014705 created "2016-06-24" @default.
• W2045014705 creator A5016041137 @default.
• W2045014705 creator A5029323921 @default.
• W2045014705 creator A5054199690 @default.
• W2045014705 date "1998-08-01" @default.
• W2045014705 modified "2023-10-17" @default.
• W2045014705 title "HYAL2, a Human Gene Expressed in Many Cells, Encodes a Lysosomal Hyaluronidase with a Novel Type of Specificity" @default.
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