Molecular Cloning and Expression of a Third Member of the Heparan Sulfate/Heparin GlcNAcN-Deacetylase/ N-Sulfotransferase Family*
- From the Division of Cellular and Molecular Medicine, Glycobiology Program, University of California, San Diego, La Jolla, California 92093-0687
Abstract
N-Deacetylation andN-sulfation of N-acetylglucosamine residues in heparan sulfate and heparin initiate a series of chemical modifications that ultimately lead to oligosaccharide sequences with specific ligand binding properties. These reactions are catalyzed by GlcNAcN-deacetylase/N-sulfotransferase (NDST), a monomeric enzyme with two catalytic activities. Two genes encoding NDST isozymes have been described, one from rat liver (NDST1) and another from murine mastocytoma (NDST2). Both isozymes are expressed in tissues in varying amounts, but their relative contribution to heparan sulfate formation in any one tissue is unknown. We now report the identification of a third member of the NDST family, designated NDST3. A full-length cDNA clone (3.2 kilobase pairs) encoding a 873-amino acid protein was obtained from a human fetal/infant brain cDNA library. Human NDST3 (hNDST3) has a nucleotide sequence homologous but not identical to hNDST1 and NDST2. The deduced amino acid sequence shows 70% and 65% amino acid identity to that of hNDST1 and NDST2, respectively. A soluble chimera of hNDST3 and protein A exhibited bothN-deacetylase and N-sulfotransferase activity, confirming its enzymatic identity. Northern blot analysis of human fetal brain poly(A)+ RNA showed a single transcript of 6.4 kilobase pairs. Reverse transcription polymerase chain reaction analysis revealed more restricted tissue expression of hNDST3 than hNDST1 and NDST2, and high levels in brain, liver, and kidney. Analysis of Chinese hamster ovary cells revealed expression of NDST1 and NDST2, but not NDST3. In a Chinese hamster ovary cell mutant exhibiting reduced N-sulfotransferase activity and reduced sulfation of heparan sulfate (Bame, K. J., and Esko, J. D. (1989)J. Biol. Chem. 264, 8059–8065), expression of NDST1 was greatly reduced, but NDST2 was expressed normally, suggesting that both enzymes are involved in heparan sulfate assembly. The discovery of multiple NDST isozymes suggests that the assembly of heparan sulfate is much complicated than previously appreciated.
Footnotes
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↵* This work was supported by NIH grants GM33063 (J.D.E.), Project 5 of Program Project Grant HL53745 (J.D.E.), and support from RIKEN (J. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF074924.
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↵‡ On leave from Laboratory of Synthetic Cellular Chemistry, RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan.
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↵§ To whom correspondence should be addressed: 9500 Gilman Dr., CMM-East 1055, La Jolla, CA 92093-0687. Tel.: 619-822-1100; Fax: 619-534-5611; E-mail: jesko{at}ucsd.edu.
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↵2 A consensus regarding the abbreviation for the proteoglycan N-sulfotransferases was reached at a recent meeting on proteoglycans. The abbreviation NDST refers specifically to the GlcNAc N-deacetylase/N-sulfotransferase involved in heparan sulfate and heparin biosynthesis. Species names precede the abbreviation using a single- or double-letter code (e.g. m for mouse, h for human, or hm for hamster) and the different isozymes contain a numeric suffix (e.g. hNDST1 refers to the human isozyme 1). NDST1 refers to the enzyme originally cloned from rat liver (4), NDST2 refers to the one originally described in murine mastocytoma (7, 9), and NDST3 refers to the one described in this paper.
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↵3 These primers were based on mouse genomic sequence homologous to hNDST3 cDNA (J. Aikawa and J. D. Esko, unpublished results). Since PCR with this set of primers using mouse brain cDNA as a template produced the 359-bp DNA fragment predicted from the genomic information, the use of this set of primers in PCR should be able to amplify a part of the Chinese hamster NDST3 cDNA.
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↵4 At the day of submission of this paper, 5 mouse and 2 rat EST clones of NDST2 were also found. A total of 26 human, 8 mouse, and 1 rat EST clones have been reported for NDST1.
- Abbreviations:
- GlcA
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d-glucuronic acid
- IdoA
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l-iduronic acid
- GlcN
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unsubstituted glucosamine
- NDST
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heparan sulfate GlcNAcN-deacetylase PAPS:GlcN N-sulfotransferase
- hNDST
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rNDST, and mNDST, human, rat, and mouse NDST, respectively
- CHO
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Chinese hamster ovary
- PAPS
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adenosine 3′-phosphate 5′-phosphosulfate
- bp
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base pair(s)
- kb
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kilobase pair(s)
- RT
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reverse transcriptase
- PCR
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polymerase chain reaction
- EST
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expressed sequence tag
- RACE
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rapid amplification of cDNA ends.
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- Received July 22, 1998.
- Revision received October 26, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
