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J Biol Chem, Vol. 274, Issue 33, 22915-22918, August 13, 1999

COMMUNICATION
Human Homolog of Caenorhabditis elegans sqv-3 Gene Is Galactosyltransferase I Involved in the Biosynthesis of the Glycosaminoglycan-Protein Linkage Region of Proteoglycans^*

Tetsuya Okajima, Kumi Yoshida^§, Tadao Kondo^¶, and Koichi Furukawa

From the  Department of Biochemistry II, Nagoya University School of Medicine, Tsurumai, Nagoya 466-0065, the ^§ School of Life Studies, Sugiyama Jogakuen University, Chikusa, Nagoya 464-0802, and the ^¶ Chemical Instrument Center, Nagoya University, Chikusa, Nagoya 464-8602, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A cDNA encoding a novel galactosyltransferase was identified based on BLAST analysis of expressed sequence tags, and the cDNA clones were isolated from a human melanoma line library. The new cDNA sequence encoded a type II membrane protein with 327 amino acid sequence and showed 38% homology to the Caenorhabditis elegans sqv-3 gene involved in the vulval invagination and oocyte development. Extracts from L cells transfected with the galactosyltransferase cDNA in an expression vector and a fusion protein with protein A exhibited marked galactosyltransferase activity specific for p-nitrophenyl--D-xylopyranoside. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis of galactosyltransferase I-deficient Chinese hamster ovary mutant pgsB-761 cells. Analysis of the enzyme product by -galactosidase digestion, mass spectroscopy, and NMR spectroscopy revealed that the reaction product was formed via -1,4 linkage, indicating that the enzyme is galactosyltransferase I (UDP-galactose:O--D-xylosylprotein 4--D-galactosyltransferase, EC 2.4.1.133) involved in the synthesis of the glycosaminoglycan-protein linkage region of proteoglycans.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proteoglycans are polyanionic molecules consisting of different core proteins and different types, numbers, and length of glycosaminoglycans (GAGs)¹ and are present not only on the cell surface but also in the extracellular matrices of various tissues (1). A wide variety of proteoglycans containing characteristic sulfated GAG chains are generated in a cell type-specific manner, and their strictly regulated expression patterns have suggested their roles in the regulation of cell proliferation/differentiation (2), tissue development and organogenesis (3), and infections (4).

The biosynthesis of the sulfated GAGs on proteoglycans is initiated by the addition of Xyl to Ser residues in the core proteins; and then the addition of two Gal residues and a GlcA residue subsequently takes place (1). Alternatively, the addition of GlcNAc or GalNAc residues to the common linkage structure leads to the formation of heparin/heparan sulfate or that of chondroitin sulfate/dermatan sulfate, respectively. The sequential transfer of individual sugars has been considered to be catalyzed by specific glycosyltransferases, and some glycosyltransferase genes responsible for individual steps have recently been isolated, i.e. glucuronyltransferase I (5, 6), GlcA/GlcNAc transferases to elongate the heparan sulfate chain (7), and a GalNAc/GlcNAc transferase to initiate this process (8).

In the present study, we isolated cDNA clones encoding human galactosyltransferase I (XGalT-1), which is involved in the biosynthesis of the common carbohydrate-protein linkage structure GlcA1,3Gal1,3Gal1,4Xyl1-O-Ser (1), based on the BLAST analysis of expressed sequence tags (EST) using the cDNA sequence of the Caenorhabditis elegans sqv-3 gene (9). We demonstrate here the substrate specificities of the cloned cDNA product and the restoration of GAG expression on the mutant CHO cells deficient in galactosyltransferase I after the introduction of the cloned cDNA. These results, as well as the structure analyses of the enzyme product, indicated that the cloned gene encodes human galactosyltransferase I.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- UDP-Gal, p-nitrophenyl--D-xylopyranoside (p-Nph--D-Xyl), p-nitrophenyl--D-galactopyranoside (p-Nph--D-Gal), p-nitrophenyl-N-acetyl--D-galactosaminide (p-Nph--D-GalNAc), p-nitrophenyl-N-acetyl--D-glucosaminide (p-Nph--D-GlcNAc), and p-nitrophenyl--D-xylopyranoside (p-Nph--D-Xyl) were purchased from Sigma. [-³²P]dCTP was from ICN (Costa Mesa, CA). Heparitinase I and II, heparinase, chondroitinase ABC, and the monoclonal antibody (mAb) for heparan sulfate (HepSS-1) were purchased from Seikagaku Corp (Tokyo, Japan).

EST Data Base Search-- Data base searches were performed with the coding sequence of the C. elegans sqv-3 gene (GenBank^TM accession no. AJ005867) using tBLASTn algorithms against the EST data base at the NCBI. Four human EST clones (GenBank^TM accession nos. AI040029, AA100869, AA442547, and T82170) were found, and overlapping sequences (nucleotides 644-1126) were obtained by reverse transcription PCR using total RNA from human colon cancer cell line Lovo as a template.

Screening of cDNA Library-- The 5' RACE product (nucleotides 29-687) prepared using the 5' RACE kit (Life Technologies, Inc.) was ³²P-labeled with a Megaprime^TM DNA labeling system (Amersham Pharmacia Biotech) and used to screen the SK-MEL-37 cDNA library. Approximately, 4 × 10⁵ recombinant clones were screened by colony hybridization. The nucleotide sequence was determined by the dideoxy termination method using an ABI PRISM^TM 310 genetic analyzer (Applied Biosystems). The newly cloned gene was designated XGalT-1 for the reasons described below.

Construction of Expression Vectors-- A cDNA fragment encoding the open reading frame of XGalT-1 was prepared by PCR using a 5' primer containing an XhoI site, 5'-CTCGAGACGATGTTCCCCTCGCGGAGG-3' (nucleotides 38-58), and a 3' primer containing a SpeI site, 5'-TCTAGAAGCTCAGCTGAATGTGCACCA-3' (nucleotides 1027-1007), and the cloned cDNA as a template. The PCR product was inserted into the XhoI and SpeI sites of the pMIKneo vector (provided by Dr. K. Maruyama, Tokyo Medical and Dental University). The truncated form of XGalT-1, lacking 53 amino acids from the amino terminus, was prepared by PCR using a 5' primer containing an EcoRI site, 5'-CAGCTCGAATTCTCTGGGGACGTGGCCCGG-3' (nucleotides 200-217), and a 3' primer containing an XhoI site, 5'-TGTCCACTCGAGTCAGCTGAATGTGCACCA-3' (nucleotides 1007-1024) and the cloned cDNA fragment as a template. The product was subcloned into the EcoRI and XhoI sites of the pCD-SA vector XGalT-1-protA.

Cell Culture-- Mouse fibroblast L cells and CHO-K1 cells were grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 7.5% FCS at 37 °C in a 5% CO₂ atmosphere. CHO mutant pgsB-761 (10) was obtained from the American Type Culture Collection and grown in F-12K medium (Life Technologies, Inc.) supplemented with 10% FCS.

Preparation of Membrane Fraction-- L cells were transiently transfected with an expression plasmid (4 µg) by the DEAE-dextran method (11). After 48 h of culture, the cells were harvested, and the membrane fraction was prepared as described (12).

Preparation of Soluble Forms of XGalT-1-- L cells (10-cm dish) were transfected with pCDSA-XGalT-1 (4 µg) by the DEAE-dextran method, and soluble forms of XGalT-1 were obtained as described (13). To prepare the sample for NMR spectroscopic analysis, the soluble enzyme was further purified using IgG-Sepharose (Amersham Pharmacia Biotech). The beads-enzyme complex was washed with and then resuspended in 100 mM MES buffer, pH 6.0.

Galactosyltransferase Assay-- The galactosyltransferase activity was determined according to Lugemwa et al. (14) with modification. The assay mixture containing 1 µl of Me₂SO, 15 mM MnCl₂, 50 mM KCl, 1% Triton X-100, 100 mM MES buffer, pH 6.0, 0.6 mM UDP-Gal, 5000 dpm/µl UDP-[¹⁴C]Gal (NEN Life Science Products), and 1 µg of the enzyme and substrates in a total volume of 25 µl. After incubation at 37 °C for 30 min, the reaction mixture was applied onto a Sep-Pak C₁₈ cartridge (Waters), and the product was eluted with 5 ml of methanol.

-Galactosidase Digestion-- One µg of [¹⁴C]Gal1-4Xyl1-p-Nph formed using XGalT-1-protA was dissolved in 200 µl of solution containing 50 mM Tris-HCl, pH 7.3, 50 mM NaCl, 20 µg of -galactosidase from Escherichia coli (Roche Molecular Biochemicals) and incubated for 7 h at 37 °C. One µg of the labeled product was also digested with 20 milliunits of diplococcal -galactosidase (Roche Molecular Biochemicals) for 23 h at 37 °C in a total volume of 100 µl of 50 mM sodium citrate buffer, pH 6.0, containing 100 mM NaCl and 100 µg/ml bovine serum albumin. The digested product was separated on a Sep-Pak C₁₈ cartridge as described above.

Purification and Identification of the Enzyme Product-- The enzyme reaction was performed in a mixture consisting of 50 µl of the enzyme-bound IgG-Sepharose, 2.7 mg of p-Nph--D-Xyl, 10 µl of Me₂SO, 15 mM MnCl₂, 50 mM KCl, 1% Triton X-100, 100 mM MES buffer, pH 6.0, and 75 mM of UDP-Gal in a final volume of 250 µl. The complete conversion of p-Nph--D-Xyl to a less migrating compound was confirmed by thin layer chromatography with a solvent system of ethanol/pyridine/n-butanol/acetate/water (100:10:10:3:30). The reaction product was purified on a Sep-Pak C₁₈ cartridge as described above and then further purified by high performance liquid chromatography (Jasco 880-PU pumps and MD915 detector) using a Develosil^TM ODS HG-5 column (4.6 × 250 mm, Nomura Chemical). Elution solvents were aqueous 10% acetonitrile (solvent A) and aqueous 30% acetonitrile (solvent B) utilizing a gradient at 40 °C. Before NMR analysis, the sample was dissolved in D₂O (99.9 atom % deuterium), dried up in vacuo, and finally dissolved in 600 µl of D₂O (99.96 atom % deuterium). ¹H NMR spectra (600 MHz) were obtained on a JEOL JMN -600 in D₂O, and one-dimensional, one-dimensional HOHAHA, and NOE difference spectra and COSY were recorded as ppm from internal t-BuOH (1.23) at 25 °C. FAB-MS data were recorded on a JEOL MStation using m-nitrobenzyl alcohol.

Flow Cytometry Analysis-- CHO pgsB-761 cells were transfected with 10 µg of plasmid DNA using LipofectAMINE^TM (Life Technologies, Inc.). Three days later, cells were analyzed by flow cytometry with mAb HepSS-1 at a dilution of 1:25 (40 µg/ml) as described (13) on a FACSCalibur with Cell Quest^TM version 3.1f software (Becton Dickinson).

[³⁵S]Sulfate Labeling and Enzyme Digestion of GAGs Synthesized in PgsB-761 Cells-- PgsB-761 cells were transfected with XGalT-1, incubated for 96 h, and then labeled with Na₂[³⁵S]SO₄ (100 µCi/ml, NEN Life Science Products) for 24 h. The labeled GAGs were isolated by protease digestion and DEAE-Sephacel chromatography (Amersham Pharmacia Biotech) according to Esko et al. (15). The isolated GAGs were treated by chondroitinase ABC (CSase) or heparitinase I and II and heparinase (HSase) and separated as described previously (16).

Northern Blotting-- Human Multiple Tissue Northern blot® was purchased from CLONTECH Laboratories and was probed with a gel-purified [-³²P]dCTP-labeled XGalT-1 cDNA or glyceraldehyde-phosphate dehydrogenase.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular Cloning of Human XGalT-1 Gene-- C. elegans sqv-3 was similar in its amino acid sequence to the 4-galactosyltransferase family of human and other vertebrates (9) and was required for vulval invagination and oocyte development (17). To find a human homolog, the NCBI Data Bank of EST cDNA clones was probed with the deduced amino acid sequence of sqv-3 cDNA. Several human partial-length cDNA clones were obtained, and the nucleotide sequence was confirmed by reverse transcription PCR (nucleotides 644-1126 in Fig. 1A) with total RNA from a human colon cancer line. Five independent cDNAs homologous to sqv-3 were obtained from a library, and three of them contained a whole open reading frame encoding a protein of 327 amino acids with a molecular mass of 37,405 daltons (Fig. 1A). It has one potential N-linked glycosylation site. The position of the AUG start codon was determined according to the Kozak consensus sequence (18). Hydropathy (19) indicated one prominent hydrophobic segment of 28 residues in length in the amino-terminal region, predicting that the protein had the type II transmembrane topology characteristic of many other glycosyltransferases cloned to date (Fig. 1B). A comparison of the primary structure of the identified cDNA and human 4GalT-1 (GenBank^TM accession no. X14085) (20) revealed that 82 of the 327 amino acids (25%) were identical (Fig. 1C). Similar results were observed between the newly cloned gene and other human 4-galactosyltransferases (4GalT-II, -III, -IV, -V, and -VI) (21-25). In contrast, this gene shares 38% identity with the sqv-3 gene, suggesting that this gene represents its human ortholog (Fig. 1C). These results indicate that the newly cloned gene product is a novel member of the 4-galactosyltransferase gene family.

View larger version (80K): [in this window] [in a new window]   Fig. 1.   Nucleotide and deduced amino acid sequences of human XGalT-1. A, the nucleotide sequence and the deduced amino acid sequence. The putative transmembrane domain is underlined, and a potential N-linked glycosylation site is boxed. B, the hydropathy calculated by the method of Kyte and Doolittle (19) with a window of 7 amino acids. C, multiple amino acid sequence alignment of human XGalT-1, C. elegans sqv-3, and human 4GalT-1 using the ClustalW program. Identical residues are boxed in black. Gray boxes indicate conserved residues, and introduced gaps are shown as hyphens.

Galactosyltransferase Activity of the Newly Cloned Enzyme-- To analyze the galactosyltransferase activity of the human sqv-3 ortholog, the expression vector of the cloned cDNA, pMIKneo-XGalT-1, was transfected into L cells, and their extracts were used for galactosyltransferase assay using a variety of acceptor substrates (Table I). Significant activity was observed with p-Nph--D-Xyl, a primer for glycosaminoglycan chain formation. No other substrates, including p-Nph--D-GlcNAc, were utilized. Almost no activity was detected in the extracts from mock-transfected cells. The apparent K_m for p-Nph--D-Xyl was 3.4 mM. These results suggest that the expressed protein is galactosyltransferase I (UDP-galactose:O--D-xylosylprotein 4--D-galactosyltransferase, EC 2.4.1.133) (26). Similar results were obtained using a soluble fusion enzyme XGalT-1-protA (data not shown).

Characterization of the Enzyme Product-- To identify the enzyme reaction products, p-Nph--D-Xyl was labeled with [¹⁴C]Gal using XGalT-1-protA and then subjected to treatment with E. coli. -galactosidase (27). More than 99% of the labeled products were digested (data not shown). Furthermore, the labeled products were also completely cleaved by diplococcal -galactosidase, which specifically digests terminal -1,4-galactosyl linkage (data not shown), indicating that XGalT-1-protA fusion protein catalyzes the galactose transfer from UDP-Gal to the acceptor in -1,4 linkage.

Structural Determination of the Enzyme Product-- To characterize the structure of the reaction product, FAB-MS was performed and revealed that the molecular weight of the product is 433 from signals at 434 m/z (M + 1) and 456 m/z (M + Na⁺), which was attributable to galactosyl products of the starting Xyl1-p-Nph. The ¹H NMR spectrum showed the p-nitrophenyl moiety and two glycosyl residues. All of the signals of the sugar moieties were assigned by 1D-HOHAHA spectra irradiating at each anomeric signal ( = 4.49 and  = 5.25) and COSY (Fig. 2). The structure of the p-nitrophenyl--xylopyranosyl moiety was confirmed by comparison with the spectrum of the starting molecule. The coupling constant of the galactosyl residue (J_1,2 = 7.7 Hz, J_2,3 = 9.9 Hz, J_3,4 = 3.7 Hz, J_4,5 = 0 Hz) indicated that the galactoside is a -pyranoside. The linkage of the galactoside was determined by NOE difference spectra. By irradiation at Gal H-1, strong NOEs were observed at Xyl H-4, Gal H-3, and Gal H-5, and also, by irradiation at Xyl H-4, Gal H-1 was strongly enhanced. Thus, the structure of the product was identified as Gal1-4Xyl1-p-Nph (28).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   1H NMR spectra of the enzyme product. A, structure of the product formed by the cloned cDNA product. B, 1H chemical shifts (ppm) and 1H-1H coupling constants (Hz) measured for galactosyl and xylosyl residues are summarized.

Restoration of GAG Synthesis by the Cloned XGalT-1 in a GAG-deficient Mutant Cell-- To confirm that this enzyme is involved in the biosynthesis of GAGs in vivo, CHO mutant pgsB-761 (galactosyltransferase I-deficient) cells were transiently transfected with pMIKneo-XGalT-1. As shown in Fig. 3A, about 50% of the transfected cells were stained with mAb HeppSS-1, whereas the mock-transfected cells were negative, indicating that the cloned cDNA actually encodes galactosyltransferase I. This result was also confirmed by ³⁵S-labeled GAGs with CSase or HSase digestion (Fig. 3B).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Restoration of GAG synthesis in pgsB-761 cells after XGalT-1 transfection. A, flow cytometry analysis of pgsB-761 cells transfected with the XGalT-1 cDNA. Cloned cDNA in pMIKneo (d) or pMIKneo alone (c) was transfected by LipofectAMINETM into pgsB-761 cells. Expression of heparan sulfate was analyzed using heparan sulfate-specific mAb HepSS-1 in flow cytometry, as described under "Experimental Procedures." CHO-K1 cells (a) and pgsB-761 cells (b) were analyzed as positive and negative controls, respectively. Thin lines are with mAb, and thick lines are controls. B, radioactivity of GAG fractions from CHO-K1, pgsB-761, and pgsB-761 cells transfected with XGalT-1. These cells were labeled with [35S]sulfate as described under "Experimental Procedures." Black bars, untreated; gray bars, treated with CSase; white bars, treated with HSase. Results are presented as cpm/µg of protein.

Expression of the XGalT-1 Gene-- Northern blotting with cDNA as a probe revealed that the XGalT-1 gene was expressed in all human tissues examined (including the heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas), and 1.8-kilobase pair transcripts were detected (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Newly cloned XGalT-1 was identified as a possible member of the 4-galactosyltransferase family in the BLAST analysis of EST using the cDNA sequence of C. elegans sqv-3. sqv-3 was identified as one of the genes possiblly encoding the components of a conserved glycosylation pathway and required for vulval invagination (9, 17). Because of the similarity of the amino acid sequence to the cloned mammalian 4-galactosyltransferases, sqv-3 seemed to be a 1,4-galactosyltransferase to create galactose 1,4-N-acetylglucosamine linkage (9). The predicted amino acid sequence of the new gene had a higher homology to sqv-3 than to other mammalian 4-galactosyltransferases, suggesting that the gene encodes a 4-galactosyltransferase distinct from known 4-galactosyltransferases involved in the synthesis of Gal1,4GlcNAc (or Glc) structures. As expected, the substrate specificity analysis revealed that the new gene encodes XGalT-1, involved in the biosynthesis of the GAG-protein linkage region of proteoglycans.

XGalT-1 was studied by Rodén's group (26, 27) and was further characterized in the study of CHO mutants defective in galactosyltransferase I (10) and in the study of clinical cases (29). The substrate specificities and storage stability were similar to the characters of the XGalT-1 enzyme analyzed in this study. The K_m value for p-Nph--D-Xyl reported (29) was also in good agreement with our results. All of these data, in addition to the results with the mutant CHO cells and those with FAB-MS and NMR, supported the identity of the new gene as human galactosyltransferase I.

Northern blotting showed a ubiquitous expression pattern, indicating the universal importance of the gene product. Since the activating and inhibitory effects of proteoglycans on cell proliferation have been elucidated (30, 31), important roles of GAGs on proteoglycans have been increasingly recognized. The phenotypes of C. elegans defective of sqv-3 gene strongly suggested the critical roles of its homolog in the morphogenesis and development of mammalian tissues. Thus, the linkage between the genetic study of C. elegans and mammalian glycobiology research would promote further understanding of the biological significance of GAGs on proteoglycans.

	ACKNOWLEDGEMENTS

We thank Dr. J. D. Esko (University of California at San Diego) for kindly providing CHO mutant pgsB-761. We also thank Dr. S. Tsuji (Riken Research Institute) for providing an expression vector pCDSA for a protein A fusion enzyme.

	FOOTNOTES

^* This work was supported by grants-in-aid for scientific research (10470029), for priority areas (10178104), and for Center of Excellence research from the Ministry of Education, Science, Sports and Culture of Japan.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^TM/EMBL Data Bank with accession number(s) AB028600.

To whom correspondence should be addressed. Tel.: 81-52-744-2070; Fax: 81-52-744-2069; E-mail: koichi@med.nagoya-u.ac.jp.

	ABBREVIATIONS

The abbreviations used are: GAG, glycosaminoglycan; CHO, Chinese hamster ovary; mAb, monoclonal antibody; PCR, polymerase chain reaction; FCS, fetal calf serum; 4-galactosyltransferase, 1,4galactosyltransferase; p-Nph--D-Xyl, p-nitrophenyl--D-xylopyranoside; XGalT-1, galactosyltransferase I (UDP-galactose:O--D-xylosylprotein 4--D-galactosyltransferase); MES, 4-morpholineethanesulfonic acid; EST, expressed sequence tag; RACE, rapid amplification of cDNA ends; NCBI, National Center for Biotechnology Information; NOE, nuclear Overhauser effect; FAB-MS, fast atom bombardment mass spectroscopy.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				H.-Y. Hwang and H. R. Horvitz The Caenorhabditis elegans vulval morphogenesis gene sqv-4 encodes a UDP-glucose dehydrogenase that is temporally and spatially regulated PNAS, October 29, 2002; 99(22): 14224 - 14229. [Abstract] [Full Text] [PDF]

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				M. Gotoh, T. Yada, T. Sato, T. Akashima, H. Iwasaki, H. Mochizuki, N. Inaba, A. Togayachi, T. Kudo, H. Watanabe, et al. Molecular Cloning and Characterization of a Novel Chondroitin Sulfate Glucuronyltransferase That Transfers Glucuronic Acid to N-Acetylgalactosamine J. Biol. Chem., October 4, 2002; 277(41): 38179 - 38188. [Abstract] [Full Text] [PDF]

				M. Gotoh, T. Sato, T. Akashima, H. Iwasaki, A. Kameyama, H. Mochizuki, T. Yada, N. Inaba, Y. Zhang, N. Kikuchi, et al. Enzymatic Synthesis of Chondroitin with a Novel Chondroitin Sulfate N-Acetylgalactosaminyltransferase That Transfers N-Acetylgalactosamine to Glucuronic Acid in Initiation and Elongation of Chondroitin Sulfate Synthesis J. Biol. Chem., October 4, 2002; 277(41): 38189 - 38196. [Abstract] [Full Text] [PDF]

				N. Vadaie, R. S. Hulinsky, and D. L. Jarvis Identification and characterization of a Drosophila melanogaster ortholog of human {beta}1,4-galactosyltransferase VII Glycobiology, October 1, 2002; 12(10): 589 - 597. [Abstract] [Full Text] [PDF]

				Z. S. Kawar, I. Van Die, and R. D. Cummings Molecular Cloning and Enzymatic Characterization of a UDP-GalNAc:GlcNAcbeta -R beta 1,4-N-Acetylgalactosaminyltransferase from Caenorhabditis elegans J. Biol. Chem., September 13, 2002; 277(38): 34924 - 34932. [Abstract] [Full Text] [PDF]

				S. Yamada, Y. Okada, M. Ueno, S. Iwata, S. S. Deepa, S. Nishimura, M. Fujita, I. Van Die, Y. Hirabayashi, and K. Sugahara Determination of the Glycosaminoglycan-Protein Linkage Region Oligosaccharide Structures of Proteoglycans from Drosophila melanogaster and Caenorhabditis elegans J. Biol. Chem., August 23, 2002; 277(35): 31877 - 31886. [Abstract] [Full Text] [PDF]