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James L. Funderburgh - One of the best experts on this subject based on the ideXlab platform.
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Keratan Sulfate biosynthesis.
IUBMB life, 2002Co-Authors: James L. FunderburghAbstract:Keratan Sulfate was originally identified as the major glycosaminoglycan of cornea but is now known to modify at least a dozen different proteins in a wide variety of tissues. Despite a large body of research documenting Keratan Sulfate structure, and an increasing interest in the biological functions of Keratan Sulfate, until recently little was known of the specific enzymes involved in Keratan Sulfate biosynthesis or of the molecular mechanisms that control Keratan Sulfate expression. In the last 2 years, however, marked progress has been achieved in identification of genes involved in Keratan Sulfate biosynthesis and in development of experimental conditions to study Keratan Sulfate secretion and control in vitro. This review summarizes current understanding of Keratan Sulfate structure and recent developments in understanding Keratan Sulfate biosynthesis.
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MINI REVIEW Keratan Sulfate: structure, biosynthesis, and function
Glycobiology, 2000Co-Authors: James L. FunderburghAbstract:The last 5 years have seen a marked increase in research on Keratan Sulfate (KS) and a concomitant increase in our understanding of the range of molecules that carry this adaptable polysaccharide. More than 15 KS-linked proteins have been identified and many of the genes encoding these have been cloned. KS-containing molecules have been identified in numerous epithelial and neural tissues in which KS expression responds to embryonic development, physiological variations, and to wound healing. A corneal cell culture system has been developed in which long-term KS biosynthesis is maintained. Progress has been made toward identification of the glycosyl- and sulfotransferases responsible for KS biosynthesis. A mouse knockout of a corneal KS-proteoglycan has provided the first experimental support for the role of KS in corneal transparency. Evidence has also been presented supporting functional roles of KS in cellular recognition of protein ligands, axonal guidance, cell motility, and in embryo implantation. These findings have served to expand the concept of what Keratan Sulfate is and the potential roles it may play in the cellular biology of diverse tissues.
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Mimecan, the 25-kDa Corneal Keratan Sulfate Proteoglycan, Is a Product of the Gene Producing Osteoglycin
Journal of Biological Chemistry, 1997Co-Authors: James L. Funderburgh, Martha L. Funderburgh, Lolita M Corpuz, Mary R. Roth, Elena S Tasheva, Gary W ConradAbstract:Abstract Bovine cornea contains three unique Keratan Sulfate proteoglycans (KSPGs), of which two (lumican and keratocan) have been characterized using molecular cloning. The gene for the third protein (KSPG25) has not been identified. This study examined the relationship between the KSPG25 protein and the gene for osteoglycin, a 12-kDa bone glycoprotein. The N-terminal amino acid sequence of KSPG25 occurs in osteoglycin cDNA cloned from bovine cornea. The osteoglycin amino acid sequence makes up the C-terminal 47% of the deduced sequence of the KSPG25 protein. Antibodies to osteoglycin reacted with intact corneal KSPG, with KSPG25 protein, and with a 36-kDa protein, distinct from lumican and keratocan. KSPG25-related proteins, not modified with Keratan Sulfate, were also detected in several connective tissues. Northern blot analysis showed mRNA transcripts of 2.4, 2.5, and 2.6 kilobases in numerous tissues with the 2.4-kilobase transcript enriched in ocular tissues. Ribonuclease protection analysis detected several protected KSPG25 mRNA fragments, suggesting alternate splicing of KSPG25 transcripts. We conclude that the full-length translation product of the gene producing osteoglycin is a corneal Keratan Sulfate proteoglycan, also present in many non-corneal tissues without Keratan Sulfate chains. The multiple size protein products of this gene appear to result from in situ proteolytic processing and/or alternative splicing of mRNA. The name mimecan is proposed for this gene and its products.
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Synthesis of Corneal Keratan Sulfate Proteoglycans by Bovine Keratocytes in Vitro
The Journal of biological chemistry, 1996Co-Authors: James L. Funderburgh, Sujatha Prakash, Martha L. Funderburgh, Mary M. Mann, Gary W ConradAbstract:Abstract Keratan Sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with Keratan Sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]Sulfate in keratocyte culture medium. This labeled KSPG contained Keratan Sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal Keratan Sulfate. Labeled Keratan Sulfate from cultures contained nonSulfated, monoSulfated, and diSulfated disaccharides that were released by digestion with endo-β-galactosidase or Keratanase II. NonSulfated disaccharides were relatively more abundant in Keratan Sulfate from culture than in corneal Keratan Sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with Keratan Sulfate chains and Sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its Keratan Sulfate chains. The availability of cell cultures secreting corneal Keratan Sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.
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molecular cloning and tissue distribution of keratocan bovine corneal Keratan Sulfate proteoglycan 37a
Journal of Biological Chemistry, 1996Co-Authors: Lolita M Corpuz, George S Bottomley, Sujatha Prakash, Martha L. Funderburgh, James L. Funderburgh, Gary W ConradAbstract:Abstract Previous studies showed that the Keratan Sulfate-containing proteoglycans of bovine corneal stroma contain three unique core proteins designated 37A, 37B, and 25 (Funderburgh, J. L., Funderburgh, M. L., Mann, M. M., and Conrad, G. W.(1991) J. Biol. Chem. 266, 14226-14231). Degenerate oligonucleotides designed from amino acid sequences of the 37A protein were used to screen a cDNA expression library from cultured bovine keratocytes. A cDNA clone coding for keratocan, a 37A protein, was isolated and sequenced. The deduced keratocan amino acid sequence is unique but related to two other Keratan Sulfate-containing proteins, lumican (the 37B core protein) and fibromodulin. These three proteins share approximately 35% amino acid identity and a number of conserved structural features. Northern hybridization and immunoblotting of tissue extracts found keratocan distribution to be more limited than that of lumican or fibromodulin. Keratocan is abundant in cornea and sclera and detected in much lesser amounts in skin, ligament, cartilage, artery, and striated muscles. Only in cornea was keratocan found to contain large, Sulfated Keratan Sulfate chains. Keratocan, like lumican, is a core protein of a major corneal proteoglycan but is present in non-corneal tissues primarily as a non-Sulfated glycoprotein.
Osami Habuchi - One of the best experts on this subject based on the ideXlab platform.
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Keratan Sulfate Gal-6-Sulfotransferase
Handbook of Glycosyltransferases and Related Genes, 2002Co-Authors: Masakazu Fukuta, Osami HabuchiAbstract:Keratan Sulfate (KS) is composed of the repeating disaccharide unit of Galβ1-4GlcNAc (poly-N-acetyllactosamine) with Sulfate groups at the 6-position of the Gal and GlcNAc residues. Most GlcNAc residues and half of the Gal residues of corneal KS are Sulfated, suggesting that sulfation of the Gal residue occurs after sulfation of the GlcNAc residue and determines the extent of sulfation of KS. Keratan Sulfate Gal-6-sulfotransferase (KSGal6ST) catalyzes the transfer of Sulfate to position 6 of the Gal residue of KS. Therefore it has been thought that KSGal6ST is responsible for producing the highly Sulfated type of KS. KSGal6ST is also able to Sulfate sialyl N-acetyllactosamine oligosaccharides. KSGal6ST may be engaged in the biosynthesis of not only KS but also glycoproteins bearing Sulfated sialyl N-acetyllactosamine or Sulfated sialyl Lewis X oligosaccharides.
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Molecular Cloning and Characterization of Human Keratan Sulfate Gal-6-Sulfotransferase
The Journal of biological chemistry, 1997Co-Authors: Masakazu Fukuta, Jouji Inazawa, Takayoshi Torii, Kaori Tsuzuki, Eriko Shimada, Osami HabuchiAbstract:We have previously cloned chondroitin 6-sulfotransferase (C6ST) cDNA from chick embryo chondrocytes. C6ST catalyzes sulfation of chondroitin, Keratan Sulfate, and sialyl N-acetyllactosamine oligosaccharides. In this study, we report the cloning and characterization of a novel sulfotransferase that catalyzes sulfation of Keratan Sulfate. This new sulfotransferase cDNA clone was obtained from a human fetal brain library by cross-hybridization with chick C6ST cDNA. The cDNA clone obtained contains a single open reading frame that predicts a type II transmembrane protein composed of 411 amino acid residues. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, Keratan Sulfate sulfotransferase activity was overexpressed, but C6ST activity was not increased over that of the control. Structural analysis of 35S-labeled glycosaminoglycan, which was formed from Keratan Sulfate by the reaction with 35S-labeled 3′-phosphoadenosine 5′-phosphoSulfate and the recombinant sulfotransferase, showed that Keratan Sulfate was Sulfated at position 6 of Gal residues. On the basis of the acceptor substrate specificity, we propose Keratan Sulfate Gal-6-sulfotransferase (KSGal6ST) for the name of the newly cloned sulfotransferase. KSGal6ST was assigned to chromosome 11p11.1–11.2 by fluorescence in situ hybridization. Among various human adult tissues, a 2.8-kilobase message of KSGal6ST was expressed mainly in the brain. When poly(A)+ RNAs from the chick embryo cornea and brain were probed with the human KSGal6ST cDNA in Northern hybridization, a clear band with about 2.8 kilobases was detected. These observations suggest that KSGal6ST may participate in the biosynthesis of Keratan Sulfate in the brain and cornea.
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Enzymatic sulfation of galactose residue of Keratan Sulfate by chondroitin 6-sulfotransferase
Glycobiology, 1996Co-Authors: Osami Habuchi, Kenji Uchimura, Yukie Hirahara, Masakazu FukutaAbstract:glycosaminoglycans formed from Keratan Sulfate and partially deSulfated Keratan Sulfate were JV-deacetylated by treatment with hydrazine/hydrazine Sulfate and then cleaved with HNO2 at pH 4, and the resulting products were reduced with NaB3H4. Analysis of the degradation products with paper chromatography and high performance liquid chromatography provided evidence that C6ST transferred Sulfate to position 6 of galactose residue which was glycosidically linked to iV-acetylglucosamine 6-Sulfate residue or to /V-acetylglucosamine residue. Northern blot analysis using poly (A)+ RNA from 12-d-old chick embryos indicated that the message of C6ST was expressed not only in the cartilage but also in the cornea in which Keratan Sulfate is actively synthesized.
Tomoya O. Akama - One of the best experts on this subject based on the ideXlab platform.
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Enzymes responsible for synthesis of corneal Keratan Sulfate glycosaminoglycans.
Journal of Biological Chemistry, 2007Co-Authors: Kazuko Kitayama, Kohji Nishida, Yasutaka Hayashida, Tomoya O. AkamaAbstract:Abstract Keratan Sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan Sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, β1,3-N-acetylglucosaminyltansferase-7 (β3GnT7) and β1,4-galactosyltransferase-4 (β4GalT4), in the production of Keratan Sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-Sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-Sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly Sulfated Keratan Sulfate by a two-step reaction in vitro with a mixture of β3GnT7/β4GalT4/CGn6ST followed by Keratan Sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly Sulfated Keratan Sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of β3GnT7 or β4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the Keratan Sulfate carbohydrate backbone.
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Enzymatic Synthesis in Vitro of the DiSulfated Disaccharide Unit of Corneal Keratan Sulfate
The Journal of biological chemistry, 2002Co-Authors: Tomoya O. Akama, Ole Hindsgaul, Anup Kumar Misra, Michiko N. FukudaAbstract:Among the enzymes of the carbohydrate sulfotransferase family, human corneal GlcNAc 6-O-sulfotransferase (hCGn6ST, also known as human GlcNAc6ST-5/GST4β) and human intestinal GlcNAc 6-O-sulfotransferase (hIGn6ST or human GlcNAc6ST-3/GST4α) are highly homologous. In the mouse, intestinal GlcNAc 6-O-sulfotransferase (mIGn6ST or mouse GlcNAc6ST-3/GST4) is the only orthologue of hCGn6ST and hIGn6ST. In the previous study, we found that hCGn6ST and mIGn6ST, but not hIGn6ST, have sulfotransferase activity to produce Keratan Sulfate (Akama, T. O., Nakayama, J., Nishida, K., Hiraoka, N., Suzuki, M., McAuliffe, J., Hindsgaul, O., Fukuda, M., and Fukuda, M. N. (2001)J. Biol. Chem. 276, 16271–16278). In this study, we analyzed the substrate specificities of these sulfotransferasesin vitro using synthetic carbohydrate substrates. We found that all three sulfotransferases can transfer Sulfate to the nonreducing terminal GlcNAc of short carbohydrate substrates. Both hCGn6ST and mIGn6ST, but not hIGn6ST, transfer Sulfate to longer carbohydrate substrates that have poly-N-acetyllactosamine structures, suggesting the involvement of hCGn6ST and mIGn6ST in production of Keratan Sulfate. To clarify further the involvement of hCGn6ST in biosynthesis of Keratan Sulfate, we reconstituted the biosynthetic pathway in vitro by sequential enzymatic treatment of a synthetic carbohydrate substrate. Using four enzymes, β1,4-galactosyltransferase-I, β1,3-N-acetylglucosaminyltransferase-2, hCGn6ST, and Keratan Sulfate Gal 6-O-sulfotransferase, we were able to synthesize in vitro a product that conformed to the basic structural unit of Keratan Sulfate. Based on these results, we propose a biosynthetic pathway for N-linked Keratan Sulfate on corneal proteoglycans.
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Human Corneal GlcNAc 6-O-Sulfotransferase and Mouse Intestinal GlcNAc 6-O-Sulfotransferase Both Produce Keratan Sulfate
Journal of Biological Chemistry, 2001Co-Authors: Tomoya O. Akama, Jun Nakayama, Kohji Nishida, Nobuyoshi Hiraoka, Misa Suzuki, Joseph Mcauliffe, Ole Hindsgaul, Minoru Fukuda, Michiko N. FukudaAbstract:Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been identified by the positional candidate approach as the gene responsible for macular corneal dystrophy (MCD). Because of its high homology to carbohydrate sulfotransferases and the presence of mutations of this gene in MCD patients who lack Sulfated Keratan Sulfate in the cornea and serum, hCGn6ST protein is thought to be a sulfotransferase that catalyzes sulfation of GlcNAc in Keratan Sulfate. In this report, we analyzed the enzymatic activity of hCGn6ST by expressing it in cultured cells. A lysate prepared from HeLa cells transfected with an intact form of hCGn6ST cDNA or culture medium from cells transfected with a secreted form of hCGn6ST cDNA showed an activity of transferring Sulfate to C-6 of GlcNAc of synthetic oligosaccharide substrates in vitro. When hCGn6ST was expressed together with human Keratan Sulfate Gal-6-sulfotransferase (hKSG6ST), HeLa cells produced highly Sulfated carbohydrate detected by an anti-Keratan Sulfate antibody 5D4. These results indicate that hCGn6ST transfers Sulfate to C-6 of GlcNAc in Keratan Sulfate. Amino acid substitutions in hCGn6ST identical to changes resulting from missense mutations found in MCD patients abolished enzymatic activity. Moreover, mouse intestinal GlcNAc 6-O-sulfotransferase had the same activity as hCGn6ST. This observation suggests that mouse intestinal GlcNAc 6-O-sulfotransferase is the orthologue of hCGn6ST and functions as a sulfotransferase to produce Keratan Sulfate in the cornea.
Masakazu Fukuta - One of the best experts on this subject based on the ideXlab platform.
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Keratan Sulfate Gal-6-Sulfotransferase
Handbook of Glycosyltransferases and Related Genes, 2002Co-Authors: Masakazu Fukuta, Osami HabuchiAbstract:Keratan Sulfate (KS) is composed of the repeating disaccharide unit of Galβ1-4GlcNAc (poly-N-acetyllactosamine) with Sulfate groups at the 6-position of the Gal and GlcNAc residues. Most GlcNAc residues and half of the Gal residues of corneal KS are Sulfated, suggesting that sulfation of the Gal residue occurs after sulfation of the GlcNAc residue and determines the extent of sulfation of KS. Keratan Sulfate Gal-6-sulfotransferase (KSGal6ST) catalyzes the transfer of Sulfate to position 6 of the Gal residue of KS. Therefore it has been thought that KSGal6ST is responsible for producing the highly Sulfated type of KS. KSGal6ST is also able to Sulfate sialyl N-acetyllactosamine oligosaccharides. KSGal6ST may be engaged in the biosynthesis of not only KS but also glycoproteins bearing Sulfated sialyl N-acetyllactosamine or Sulfated sialyl Lewis X oligosaccharides.
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Molecular Cloning and Characterization of Human Keratan Sulfate Gal-6-Sulfotransferase
The Journal of biological chemistry, 1997Co-Authors: Masakazu Fukuta, Jouji Inazawa, Takayoshi Torii, Kaori Tsuzuki, Eriko Shimada, Osami HabuchiAbstract:We have previously cloned chondroitin 6-sulfotransferase (C6ST) cDNA from chick embryo chondrocytes. C6ST catalyzes sulfation of chondroitin, Keratan Sulfate, and sialyl N-acetyllactosamine oligosaccharides. In this study, we report the cloning and characterization of a novel sulfotransferase that catalyzes sulfation of Keratan Sulfate. This new sulfotransferase cDNA clone was obtained from a human fetal brain library by cross-hybridization with chick C6ST cDNA. The cDNA clone obtained contains a single open reading frame that predicts a type II transmembrane protein composed of 411 amino acid residues. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, Keratan Sulfate sulfotransferase activity was overexpressed, but C6ST activity was not increased over that of the control. Structural analysis of 35S-labeled glycosaminoglycan, which was formed from Keratan Sulfate by the reaction with 35S-labeled 3′-phosphoadenosine 5′-phosphoSulfate and the recombinant sulfotransferase, showed that Keratan Sulfate was Sulfated at position 6 of Gal residues. On the basis of the acceptor substrate specificity, we propose Keratan Sulfate Gal-6-sulfotransferase (KSGal6ST) for the name of the newly cloned sulfotransferase. KSGal6ST was assigned to chromosome 11p11.1–11.2 by fluorescence in situ hybridization. Among various human adult tissues, a 2.8-kilobase message of KSGal6ST was expressed mainly in the brain. When poly(A)+ RNAs from the chick embryo cornea and brain were probed with the human KSGal6ST cDNA in Northern hybridization, a clear band with about 2.8 kilobases was detected. These observations suggest that KSGal6ST may participate in the biosynthesis of Keratan Sulfate in the brain and cornea.
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Enzymatic sulfation of galactose residue of Keratan Sulfate by chondroitin 6-sulfotransferase
Glycobiology, 1996Co-Authors: Osami Habuchi, Kenji Uchimura, Yukie Hirahara, Masakazu FukutaAbstract:glycosaminoglycans formed from Keratan Sulfate and partially deSulfated Keratan Sulfate were JV-deacetylated by treatment with hydrazine/hydrazine Sulfate and then cleaved with HNO2 at pH 4, and the resulting products were reduced with NaB3H4. Analysis of the degradation products with paper chromatography and high performance liquid chromatography provided evidence that C6ST transferred Sulfate to position 6 of galactose residue which was glycosidically linked to iV-acetylglucosamine 6-Sulfate residue or to /V-acetylglucosamine residue. Northern blot analysis using poly (A)+ RNA from 12-d-old chick embryos indicated that the message of C6ST was expressed not only in the cartilage but also in the cornea in which Keratan Sulfate is actively synthesized.
Gary W Conrad - One of the best experts on this subject based on the ideXlab platform.
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analysis of Keratan Sulfate oligosaccharides by electrospray ionization tandem mass spectrometry
Analytical Chemistry, 2005Co-Authors: Yuntao Zhang, Elena S Tasheva, Yutaka Kariya, Abigail H Conrad, Gary W ConradAbstract:Keratan Sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked β-(1−4) and β-(1−3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify Keratan Sulfate oligosaccharides. Two nonSulfated disaccharide isomers and two monoSulfated disaccharide isomers were distinguished through MS/MS. In MS1 spectra of multiply Sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of Sulfates. Subsequent MS2 and MS3 spectra of mono-, di-, tri-, and tetraSulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolatio...
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Mimecan, the 25-kDa Corneal Keratan Sulfate Proteoglycan, Is a Product of the Gene Producing Osteoglycin
Journal of Biological Chemistry, 1997Co-Authors: James L. Funderburgh, Martha L. Funderburgh, Lolita M Corpuz, Mary R. Roth, Elena S Tasheva, Gary W ConradAbstract:Abstract Bovine cornea contains three unique Keratan Sulfate proteoglycans (KSPGs), of which two (lumican and keratocan) have been characterized using molecular cloning. The gene for the third protein (KSPG25) has not been identified. This study examined the relationship between the KSPG25 protein and the gene for osteoglycin, a 12-kDa bone glycoprotein. The N-terminal amino acid sequence of KSPG25 occurs in osteoglycin cDNA cloned from bovine cornea. The osteoglycin amino acid sequence makes up the C-terminal 47% of the deduced sequence of the KSPG25 protein. Antibodies to osteoglycin reacted with intact corneal KSPG, with KSPG25 protein, and with a 36-kDa protein, distinct from lumican and keratocan. KSPG25-related proteins, not modified with Keratan Sulfate, were also detected in several connective tissues. Northern blot analysis showed mRNA transcripts of 2.4, 2.5, and 2.6 kilobases in numerous tissues with the 2.4-kilobase transcript enriched in ocular tissues. Ribonuclease protection analysis detected several protected KSPG25 mRNA fragments, suggesting alternate splicing of KSPG25 transcripts. We conclude that the full-length translation product of the gene producing osteoglycin is a corneal Keratan Sulfate proteoglycan, also present in many non-corneal tissues without Keratan Sulfate chains. The multiple size protein products of this gene appear to result from in situ proteolytic processing and/or alternative splicing of mRNA. The name mimecan is proposed for this gene and its products.
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Synthesis of Corneal Keratan Sulfate Proteoglycans by Bovine Keratocytes in Vitro
The Journal of biological chemistry, 1996Co-Authors: James L. Funderburgh, Sujatha Prakash, Martha L. Funderburgh, Mary M. Mann, Gary W ConradAbstract:Abstract Keratan Sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with Keratan Sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]Sulfate in keratocyte culture medium. This labeled KSPG contained Keratan Sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal Keratan Sulfate. Labeled Keratan Sulfate from cultures contained nonSulfated, monoSulfated, and diSulfated disaccharides that were released by digestion with endo-β-galactosidase or Keratanase II. NonSulfated disaccharides were relatively more abundant in Keratan Sulfate from culture than in corneal Keratan Sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with Keratan Sulfate chains and Sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its Keratan Sulfate chains. The availability of cell cultures secreting corneal Keratan Sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.
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molecular cloning and tissue distribution of keratocan bovine corneal Keratan Sulfate proteoglycan 37a
Journal of Biological Chemistry, 1996Co-Authors: Lolita M Corpuz, George S Bottomley, Sujatha Prakash, Martha L. Funderburgh, James L. Funderburgh, Gary W ConradAbstract:Abstract Previous studies showed that the Keratan Sulfate-containing proteoglycans of bovine corneal stroma contain three unique core proteins designated 37A, 37B, and 25 (Funderburgh, J. L., Funderburgh, M. L., Mann, M. M., and Conrad, G. W.(1991) J. Biol. Chem. 266, 14226-14231). Degenerate oligonucleotides designed from amino acid sequences of the 37A protein were used to screen a cDNA expression library from cultured bovine keratocytes. A cDNA clone coding for keratocan, a 37A protein, was isolated and sequenced. The deduced keratocan amino acid sequence is unique but related to two other Keratan Sulfate-containing proteins, lumican (the 37B core protein) and fibromodulin. These three proteins share approximately 35% amino acid identity and a number of conserved structural features. Northern hybridization and immunoblotting of tissue extracts found keratocan distribution to be more limited than that of lumican or fibromodulin. Keratocan is abundant in cornea and sclera and detected in much lesser amounts in skin, ligament, cartilage, artery, and striated muscles. Only in cornea was keratocan found to contain large, Sulfated Keratan Sulfate chains. Keratocan, like lumican, is a core protein of a major corneal proteoglycan but is present in non-corneal tissues primarily as a non-Sulfated glycoprotein.
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Unique glycosylation of three Keratan Sulfate proteoglycan isoforms.
Journal of Biological Chemistry, 1991Co-Authors: James L. Funderburgh, Martha L. Funderburgh, Mary M. Mann, Gary W ConradAbstract:Abstract Recent work demonstrates isoforms of bovine corneal Keratan Sulfate proteoglycan containing structurally unique core proteins of 25 and 37 kDa (Funderburgh, J., and Conrad, G. (1990) J. Biol. Chem. 265, 8297-8303). In the current study, two forms (37A and 37B) of the 37-kDa protein were separated by ion-exchange chromatography after removal of Keratan Sulfate with endo-beta-galactosidase. Keratan Sulfate linkage sites in core proteins were labeled with UDP-[3H]galactose using galactosyltransferase. Labeled proteins were separated by sodium dodecyl Sulfate-polyacrylamide gel electrophoresis and analyzed by tryptic digestion and reversed-phase chromatography. The 37A protein has three Keratan Sulfate-linkage sites, and the 37B and 25-kDa proteins each contain one linkage site. Reversed-phase tryptic maps of the three proteins differed in total peptide profile and in glycosylated peptides labeled with periodate-[3H]-NaBH4. Tryptic mapping of the two 37-kDa isoforms after deglycosylation showed differences in total tryptic peptides, in peptides labeled with [14C]iodoacetic acid, and in peptides recognized by antibodies to a mixture of the 37-kDa cores. Antibody to a synthetic peptide with N-terminal sequence obtained from mixed 37-kDa cores reacted exclusively with the 37B isoform. These results show that bovine corneal Keratan Sulfate proteoglycan has three different core proteins each with distinct glycosylation and unique primary structure.
