J Biol Chem, Vol. 274, Issue 37, 26165-26171, September 10, 1999

Cloning and Expression of a Proteoglycan UDP-Galactose:-Xylose 1,4-Galactosyltransferase I
A SEVENTH MEMBER OF THE HUMAN 4-GALACTOSYLTRANSFERASE GENE FAMILY^*

Raquel Almeida^§, Steven B. Levery^¶, Ulla Mandel, Hans Kresse, Tilo Schwientek, Eric P. Bennett, and Henrik Clausen^**

From the  School of Dentistry, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen N, Denmark, the ^§ Institute of Molecular Pathology and Immunology of University of Porto, Rua Dr. R. Frias s/n, 4200 Porto, Portugal, the ^¶ University of Georgia, Complex Carbohydrate Research Center, Athens, Georgia 30602, and the  Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, 48129 Münster, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A seventh member of the human 4-galactosyltransferase family, 4Gal-T7, was identified by BLAST analysis of expressed sequence tags. The coding region of 4Gal-T7 depicts a type II transmembrane protein with sequence similarity to 4-galactosyltransferases, but the sequence was distinct in known motifs and did not contain the cysteine residues conserved in the other six members of the 4Gal-T family. The genomic organization of 4Gal-T7 was different from previous 4Gal-Ts. Expression of 4Gal-T7 in insect cells showed that the gene product had 1,4-galactosyltransferase activity with -xylosides, and the linkage formed was Gal1-4Xyl. Thus, 4Gal-T7 represents galactosyltransferase I enzyme (xylosylprotein 1,4-galactosyltransferase; EC 2.4.1.133), which attaches the first galactose in the proteoglycan linkage region GlcA1-3Gal1-3Gal1-4Xyl1-O-Ser. Sequence analysis of 4Gal-T7 from a fibroblast cell line of a patient with a progeroid syndrome and signs of the Ehlers-Danlos syndrome, previously shown to exhibit reduced galactosyltransferase I activity (Quentin, E., Gladen, A., Rodén, L., and Kresse, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1342-1346), revealed two inherited allelic variants, 4Gal-T7^186D and 4Gal-T7^206P, each with a single missense substitution in the putative catalytic domain of the enzyme. 4Gal-T7^186D exhibited a 4-fold elevated K_m for the donor substrate, whereas essentially no activity was demonstrated with 4Gal-T7^206P. Molecular cloning of 4Gal-T7 should facilitate general studies of its pathogenic role in progeroid syndromes and connective tissue disorders with affected proteoglycan biosynthesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Six members of a family of human UDP-galactose:-N-acetylglucosamine/-glucosylceramide 1,4-galactosyltransferases (4Gal-Ts)¹ have previously been characterized (1-7). These six 4Gal-Ts catalyze biosynthesis of Gal1-4GlcNAc and/or Gal1-4Glc linkages in different glycoconjugates and free saccharides (for a review see Ref. 8). The six 4Gal-Ts have highly conserved sequence motifs in the putative catalytic domain including four conserved cysteine residues. The genomic organization of the first four genes is similar and includes conservation of spacing for five intron/exon boundaries in the coding regions (4, 7, 9, 10). This suggests that these genes arose late in evolutionary terms as a result of gene duplication and subsequent sequence divergence. Detailed analysis of the kinetic properties of these enzymes clearly show that each has a distinct function in biosynthesis of different glycoconjugates and saccharide structures, but in accordance with their close evolutionary relationships the linkages formed are similar.

In the present study, a seventh homologue of the 4Gal-T gene family was characterized. The gene was identified by sequence analysis of the EST data base. The coding region of the novel gene, designated 4Gal-T7, exhibited distinct substitutions in the sequence motifs highly conserved among 4Gal-T1 to 4Gal-T6. Notably, none of the four cysteines conserved among other 4Gal-Ts were found in the 4Gal-T7 sequence. It was predicted that the enzymatic properties of 4Gal-T7 were different from other 4Gal-Ts. Analysis of the substrate specificity of recombinant 4Gal-T7 revealed that this enzyme formed the Gal1-4Xyl1-R linkage found in the linkage region of proteoglycans (GlcA1-3Gal1-3Gal1-4Xyl1-O-Ser). 4Gal-T7 was proposed to encode a galactosyltransferase I (xylosylprotein 1,4-galactosyltransferase; EC 2.4.1.133) gene (11-13).

Quentin-Hoffmann et al. (14, 15) showed that partial inactivation of galactosyltransferase I represented the primary defect in one patient with progeroidal appearance and symptoms of the Ehlers-Danlos syndrome. As a consequence of the enzyme deficiency, only about half of the core proteins of the small proteoglycans decorin and biglycan were linked with glycosaminoglycan chains (16),² whereas no abnormality in the biosynthesis of large dermatan sulfate proteoglycans and of heparan sulfate proteoglycans could be observed (14). We sequenced the 4Gal-T7 coding region of DNA from fibroblasts established from this patient and his family. Two alleles, 4Gal-T7^186D and 4Gal-T7^206P, were identified in the affected patient, and each allele was shown to be derived from different parents. Expression of the variant alleles showed that one exhibited a significantly higher K_m for the donor substrate and the other was inactive. The results show that 4Gal-T7 represents one galactosyltransferase I that is involved in proteoglycan synthesis. Identification of the molecular basis of the genetic defect in the progeroid patient with signs of the Ehlers-Danlos syndrome opens the possibility of further studies on the role of this gene in progeroid syndromes and connective tissue disorders.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification and Cloning of 4Gal-T7-- tBLASTn analysis of the dbEST data base at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD) with sequences from human 4Gal-T1 to 4Gal-T6, performed as described previously (4, 8), revealed several ESTs covering a total of 931 base pairs of the 3' coding sequence of 4Gal-T7. Additional sequence was obtained by 5'-rapid amplification of cDNA ends with a fetal brain cDNA library (CLONTECH) using antisense primer EBER1218 (5'-CTGAAGTGGTCCACCTGGTTG-3') and sequencing on PAC genomic DNA. ESTs from 4Gal-T7 are represented in two Unigene clusters, Hs.54702 and Hs.45208, where the latter originate from priming in the second intron of the five introns identified in 4Gal-T7. Hs.45208 was mapped to 5q35.1-5q35.3 and was flanked by D5S498 and D5S408 (stSG40105, 184.7-195.8 cM). The complete compiled cDNA sequence was confirmed by sequencing of a PAC genomic DNA clone. A human PAC genomic library (Genome Systems) was screened using the primer pairs EBER1207 (5'-CAGAGAACGGGTCTGTCACAGG-3') and EBER1215 (5'-GATGTGGTGCCGGATCTTCTT-3'). Three clones for 4Gal-T7 (99/C24, 143/B4, and 222/H10) were obtained from Genome Systems Inc. Intron/exon boundaries were determined by comparison with the cDNA sequence, optimizing for the gt/ag rule (17).

Expression of 4Gal-T7 in Insect Cells-- Expression constructs designed to encode the full coding sequence and a secreted construct encoding amino acid residues 63-327 of 4Gal-T7 were prepared by reverse transcription-PCR with fetal brain mRNA. Products were cloned initially into pBluescript KS+ (Stratagene) and subsequently into pVL1393 or pAcGP67 (Pharmingen). Expression constructs of 4Gal-T7 variants were prepared similarly using mRNA from fibroblasts of a patient with galactosyltransferase I deficiency (14). Plasmids pVL-4Gal-T7-full and pAcGP67-4Gal-T7-sol were co-transfected with Baculo-Gold^TM DNA (Pharmingen) and virus amplified as described previously (18). The kinetic properties were determined with the secreted enzyme expressed in Sf9 or High Five^TM cells. Purification of the secreted enzyme from High Five cells was performed by consecutive chromatographic steps on DEAE or Amberlite and S-Sepharose as described previously (19). Standard assays were performed in 50-µl reaction mixtures containing 25 mM cacodylate (pH 7.0), 40 mM MnCl₂, 0.25% Triton X-100, 100 µM UDP-[¹⁴C]-Gal (2,000 cpm/nmol) (Amersham Pharmacia Biotech), and the indicated concentrations of acceptor substrates (Sigma and Dextra Laboratories Ltd.) (see Table I for structures). The full-length construct was assayed with 1% Triton X-100 homogenates of cells twice washed in phosphate-buffered saline. Assays for determination of K_m for the acceptor substrates were performed with semi-purified enzyme in the standard reaction mixture modified to include 200 µM UDP-[¹⁴C]-Gal for 4Gal-T7 and 400 µM for 4Gal-T7_186D. Assays for donor substrate K_m were performed with 2.0 mM MeUmb--Xylose.

For product characterization 5 mg of MeUmb--Xylose were glycosylated to completion with semipurified 4Gal-T7, using thin layer chromatography to monitor reaction progress. The reaction product was purified on an octadecyl-silica cartridge (Bakerbond, J. T. Baker), deuterium exchanged by repeated lyophilization from D₂O, and then dissolved in 0.5 ml of D₂O for NMR analysis. One-dimensional ¹H NMR, two-dimensional ¹H-¹H TOCSY, and ¹H-detected, ¹³C-decoupled, phase-sensitive gradient ¹³C-¹H HSQC and HMBC experiments were performed as described previously (Ref. 20 and references cited therein) on a Varian Unity Inova 600 MHz spectrometer using standard acquisition software available in the Varian VNMR software package. One-dimensional reference ¹³C NMR spectra were acquired using direct detection on a Varian Unity Inova 500 MHz spectrometer. A saturated solution of Xyl17MU was prepared for NMR analysis in similar manner, and spectra were acquired under identical conditions for comparison. Chemical shifts are referenced to internal acetone (2.225 and 30.00 ppm for ¹H and ¹³C, respectively).

Northern Analysis-- The cDNA fragment of soluble 4Gal-T7 was used as a probe. The probe was random priming labeled using [³²P]dCTP and an Strip-EZ DNA labeling kit (Ambion). A multiple human tissue Northern blot, MTN I (CLONTECH), was probed as described previously (7).

Monoclonal Antibody-- A purified secreted form of 4Gal-T7 was used for immunizing BALB/c mice, and monoclonal antibodies were selected and characterized by immunocytology on Sf9 cells infected with various 4Gal-transferase expression constructs, as described previously (21). The specificity was further evaluated by SDS-polyacrylamide gel electrophoresis Western blot analysis using precast 8-25% gradient gels and the Phast system^TM (Amersham Pharmacia Biotech).

Analysis of 4Gal-T7 Gene in a Family with a Genetic Defect in Galactosyltransferase I-- Skin fibroblast cell lines from one affected patient, the parents, and two siblings were established and grown as described previously (14). mRNA was isolated and reverse transcription-PCR products were directly sequenced and/or subcloned and sequenced. The identified sequence variations were confirmed by direct sequencing PCR products obtained from genomic DNA. One missense mutation (557CA) was also identified by restriction digestion (HinfI) of a PCR product from the mutant allele.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification and Cloning of Human 4Gal-T7-- The EST cloning strategy produced a novel gene with an open reading frame of 327 amino acids exhibiting sequence similarity to members of the 4Gal-T gene family. The predicted coding region of 4Gal-T7 has a single initiation codon in agreement with Kozak's rule (22), which precedes a sequence encoding a potential hydrophobic transmembrane segment (Fig. 1A) (DNA sequence available in GenBank^TM). 4Gal-T7 is predicted to be a type II transmembrane glycoprotein with a N-terminal cytoplasmic domain of 28 residues, a transmembrane segment of 30 residues, and a stem region and catalytic domain of 269 residues. The calculated molecular weight of the protein derived from the full coding is 37,404, and proteolytically cleaved secreted forms is predicted to be less than 31,065 (calculated from Arg⁵⁹ immediately after the hydrophobic transmembrane signal sequence). Multiple sequence alignment of the seven human 4Gal-transferases (Fig. 1A) shows that the sequence of 4Gal-T7 is distinct from other 4Gal-Ts in two potentially significant regions: the major conserved sequence motif (WGWGG/REDDD/E) is not conserved in two positions, and none of the four cysteine residues conserved among the first six 4Gal-Ts are conserved in 4Gal-T7. 4Gal-T7 has a single N-linked glycosylation consensus site, which is different from a site conserved among 4Gal-T2 to 4Gal-T6.

View larger version (74K): [in this window] [in a new window]   Fig. 1.   A, multiple sequence analysis (ClustalW) of human 4Gal-Ts and two C. elegans homologues. Introduced gaps are shown as hyphens, and aligned identical residues are boxed (black for all sequences, dark gray for eight and seven sequences, and light gray for six and five sequences). The putative transmembrane domain of 4Gal-T7 is underlined with a single line. The amino acid substitutions in 4Gal-T7186D and 4Gal-T7206P are indicated above the 4Gal-T7 sequence. Positions of intron/exon boundaries in 4Gal-T7 are indicated by solid arrows below the amino acid sequence, and the conserved boundaries in 4Gal-T1 to 4Gal-T4 indicated by solid arrows below the amino acid sequence of 4Gal-T1. Intron/exon boundaries in the two C. elegans homologues are indicated by solid arrows below the respective amino acid sequences. The genomic organizations of 4Gal-T5 and 4Gal-T6 are not completed. B, phylogenetic tree of human and two C. elegans 4Gal-T homologues. The phylogenetic tree (unrooted) was produced with DNASIS software (Hitachi) based on the ClustalW multiple sequence alignment analysis presented in A using the full coding sequences of the nine genes.

The coding region of 4Gal-T7 was found in six exons (base pairs 1-50, 51-413, 414-639, 640-723, 724-828, and 829-984) similar to other 4Gal-Ts. However, in contrast to the five intron/exon boundaries conserved in 4Gal-T1 to 4Gal-T4, none of the five boundaries of 4Gal-T7 appears to be conserved (Fig. 1A). The 3' ESTs assembled in Hs.45208 were found in the second intron sequence of 4Gal-T7 and were linked to chromosome 5q35.1 to 5q35.3.

Expression of 4Gal-T7-- Expression of full coding or secreted constructs of 4Gal-T7 in insect cells resulted in marked increase in galactosyltransferase activity with a number of Xyl containing acceptor substrates, compared with uninfected cells or cells infected with a control construct (Table I). The best acceptor substrate identified was -MeUmb-Xyl, for which the K_m was estimated at 0.89 ± 0.29 mM. The K_m for UDP-Gal was 56 ± 12 µM using -MeUmb-Xyl as an acceptor. Low activity was observed with GlcNAc acceptors, but there was no activity with other mono- or disaccharide substrates tested. Structural characterization by ¹H NMR of the product formed with -MeUmb-Xyl showed that the 4Gal-T7 forms the Gal1-4Xyl1-R linkage (Table II and Fig. 2). Comparison of a one-dimensional ¹H NMR spectrum of the product (Fig. 2A) with that of substrate obtained under similar conditions (in D₂O, 25 °C; not shown) clearly showed an additional H-1 resonance (4.512 ppm) from a sugar residue linked in the -configuration (³J_{1, 2} = 7-9 Hz). We did not find NMR data for the specific expected product in the literature or in glycoconjugate NMR data bases. The substantial anisotropic effects of the 4-methylumbelliferyl group obviates direct comparison of chemical shift data with those of other glycosides, e.g. linked to L-serine (23). Thus, a de novo sequence analysis of the product was undertaken by consecutive application of two-dimensional ¹H-¹H TOCSY, ¹H-¹³C HSQC, and ¹H-¹³C HMBC NMR experiments (for a review of this strategy, see Ref. 24). Although ¹H chemical shift data were available for the Xyl17MeUmb substrate (25), these were acquired in dimethyl sulfoxide-d₆, which is known to alter proton shifts when compared with D₂O solutions (26). Therefore, direct comparison of spectral data for the product were derived from an additional series of NMR experiments on the substrate glycoside dissolved in D₂O. Thus, all ¹H and ¹³C resonances were uniquely and unambiguously assigned by the TOCSY and HSQC experiments (Table II). The H-5ax and H-5eq resonances for -Xyl were assigned on the basis of their ³J_4,5 coupling constants; the trends for ¹H resonances and coupling constants were similar to those observed for the corresponding L-serine glycosides (24); and the linkages were unambiguously established by observation of interglycosidic H1C1O1Cx and C1O1CxHx correlations in the HMBC spectrum. As shown in Fig. 2B, evidence of the newly formed Gal14Xyl linkage in the product is clearly demonstrated by strong cross-peaks correlating -Gal H-1 (4.512 ppm) with -Xyl C4 (76.02 ppm) and the corresponding -Gal C-1 (101.60 ppm) with the -Xyl H-4 resonance (3.972 ppm). Because -Xyl H-4 is completely resolved and unique in its coupling pattern with H-3, H-5eq, and H-5ax and because there are no instances of strong coupling between any of the -Xyl ring proton resonances that might otherwise create uncertainty in their assignments, the latter correlation in particular renders assignment of the linkage unambiguous. Consistent with this, the -Xyl C-4 resonance shows a substantial downfield glycosylation-induced shift ( = 7.27 ppm) that is unique when comparing product to substrate, whereas C-3 and C-5 exhibit small upfield shift changes, as expected (27). All of the proton resonances of the -Xyl residue exhibit downfield glycosylation-induced shifts: H-4 was the largest ( = 0.231 ppm). These results confirm the linkage structure of the product as 1-4.

View this table: [in this window] [in a new window]   Table I Acceptor substrate specificities of the secreted forms of 4Gal-T7 variants Background values obtained with uninfected cells or cells infected with an irrelevant construct were subtracted. The background was not higher than 0.4 nmol/min/ml. Me, methyl; Nph, nitrophenyl; Bzl, benzyl; ND, not determined.

View this table: [in this window] [in a new window]   Table II 1II, 13C chemical shifts (ppm) and 1H-1H coupling constants (Hz) for XylMU substrate and biosynthetic Gal4XylMU product in D2O at 25 °C Chemical shifts are referenced to internal acetone (2.225 and 30.00 ppm for 1II and 13C, respectively).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   A, sections of a one-dimensional 1H NMR spectrum of the 4Gal-T7 product, Gal14Xyl17MU, showing all nonexchangeable monosaccharide ring methine and exocyclic methylene resonances. Residue designations for -Gal (Gal4-) and -Xyl (Xyl-) are followed by proton designations (1-6 and 6' and 1-5ax/eq, for the two residues, respectively). B, section of the 1H-detected 1H-13C HMBC spectrum showing interglycosidic H1C1O1Cx and C1O1CxHx correlations. Cross-peaks marked by ovals or cross-hairs. The unmarked cross-peaks are all intraresidue correlations.

Expression Pattern of 4Gal-T7-- Northern analysis with mRNA from eight human adult organs showed a ubiquitous pattern of expression for 4Gal-T7 (Fig. 3). The transcript size of 4Gal-T7 was approximately 2 kilobases.

View larger version (84K): [in this window] [in a new window]   Fig. 3.   Northern blot analysis of human tissues. A multiple human Northern blot, MTNI, from CLONTECH was probed with the secreted expression construct of 4Gal-T7. The size of the labeled transcript is approximately 2 kilobases, but the transcript in skeletal muscle may be larger (approximately 2.2 kilobases).

4Gal-T7 Variants in a Patient with a Defect in Galactosyltransferase I and Proteoglycan Biosynthesis-- Sequence analysis of 4Gal-T7 mRNA and DNA in a patient with defective galactosyltransferase I activity revealed two missense mutations in coding exon III, C⁵⁵⁷  A and T⁶¹⁷  C, which result in changes in amino acid sequence, respectively, Ala¹⁸⁶  Asp and Leu²⁰⁶  Pro (Figs. 1 and 4). A genotyping strategy involving selective restriction digestion with HinfI of a PCR product confirmed the C⁵⁵⁷  A mutation (not shown). Both substitutions are in the putative catalytic domain. The Ala¹⁸⁶  Asp substitution results in introduction of an acidic residue in a fairly conserved position among 4Gal-Ts (Ala/Val/Ser) (Fig. 1A). The Leu²⁰⁶  Pro substitution results in a nonconservative change of a fully conserved Leu residue (Fig. 1A). As shown in Fig. 4, sequence analysis of the patient's family confirmed that the mother was heterozygous for the Ala¹⁸⁶  Asp allele and the father heterozygous for the Leu²⁰⁶  Pro allele. Two siblings were also heterozygous for one or the other variant alleles. The sibling with the Leu²⁰⁶  Pro allele was previously judged to be heterozygous based on analysis of galactosyltransferase I activity.³ Expression of the secreted forms of the variant alleles revealed that 4Gal-T7^186D was active (Table I), but the K_m for the donor substrate was elevated (K_m 230 ± 64 µM). The K_m for the acceptor MeUmb--Xyl was comparable with wild type (K_m 0.54 ± 0.10 mM). In contrast, expression of the 4Gal-T7^206P variant did not result in significant activity in the supernatant or in cell extracts of infected insect cells (Table I). SDS-polyacrylamide gel electrophoresis Western blot analysis with a monoclonal antibody to human 4Gal-T7 confirmed that proteins of appropriate sizes were expressed in all cases (Fig. 5). The monoclonal antibody, URH1(2C3) (IgG1), reacted specifically with cells infected with full coding or secreted constructs of 4Gal-T7 by immunocytology (not shown), and in Western blot analysis only one band corresponding to approximately 35.000 was detected in extracts of insect cells if these were infected with 4Gal-T7 constructs (Fig. 5).

View larger version (53K): [in this window] [in a new window]   Fig. 4.   Sequence analysis of position C557 and T617 in a patient with a progeroid syndrome and signs of the Ehlers-Danlos syndrome, four family members, and a control blood donor. Part of coding exon III was PCR amplified, and the product was directly sequenced. I, sequence window of position 557. II, sequence window of position 617. The patient is heterozygous for both identified mutations (557CA and 617TC). The mother and sibling 2 are heterozygous for one mutation (557CA), whereas the father and sibling 1 are heterozygous for the other mutation (617TC).

View larger version (65K): [in this window] [in a new window]   Fig. 5.   Western blot analysis of the expression of 4Gal-T7186D and 4Gal-T7206P in Sf9 cells. A, Coomassie stain. B, immunoblotting with monoclonal anti-4Gal-T7 antibody, URH1. Cells were harvested 60 h postinfection. The band labeled in cells infected with pAcGP67-4Gal-T7-sol variants is approximately 35,000, which is in agreement with the calculated mass of the recombinant secreted protein if the single N-glycosylation site is utilized.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 4Gal-T7 gene characterized in this report encodes a 4-galactosyltransferase with galactosyltransferase I activity. Such an enzyme is required for the synthesis of the tetrasaccharide linkage region of proteoglycans (11-13, 28). The finding that two allelic variants of the 4Gal-T7 gene in a patient previously shown to exhibit a defect in galactosyltransferase I activity and in the biosynthesis of small chondroitin/dermatan sulfate proteoglycans had reduced or impaired functions strongly indicates that 4Gal-T7 is one of potentially multiple galactosyltransferase I genes. Furthermore, 4Gal-T7 is the galactosyltransferase I gene that is functionally important for small proteoglycan biosynthesis in skin fibroblasts.

Given the number of genes encoding 4-galactosyltransferases transferring galactose to GlcNAc and Glc and considering the multitude of enzymes involved in catalyzing specific steps in the biosynthesis of glycosaminoglycans (e.g. glucosamine:3-O-sulfotransferases (29)), we hypothesize that additional Xyl 4-galactosyltransferase genes exist. This is in agreement with the finding that in the patient fibroblasts no abnormality in the biosynthesis of versican and of heparan sulfate proteoglycans had been found (14). However, it is also possible that only a single galactosyltransferase I gene exists and that the active mutant allele, 4Gal-T7^186D, has differential catalytic efficiency for transferring galactose to proteoglycans with different densities of glycosaminoglycan attachment sites. Independent studies on chemically mutagenized Chinese hamster ovary cells are in agreement with the proposal that either only a single galactosyltransferase I exists or that, alternatively, a single auxiliary protein for these enzymes is required. Interestingly, the mutant Chinese hamster ovary cells, which exhibited only about 2% of the normal level of galactosyltransferase I activity, could prime glycosaminoglycan synthesis on exogenously added -xylosides (30), although this activity may stem from Glc(NAc) 4GalTs (7, 31).

The catalytic properties of 4Gal-T7 resemble those of a partially purified galactosyltransferase I activity derived from embryonic chick cartilage (11). Both enzyme activities showed a K_m for UDP-Gal of approximately 50 µM. Analysis of K_m for the donor substrate with cell extracts appear to give higher K_m of 100 µM (12) or 170 µM (14). Apparent K_m values for different acceptor substrates tested vary around 0.5-6 mM (14). The properties of the recombinant allelic variants of 4Gal-T7, 4Gal-T7^186D and 4Gal-T7^206P were not in agreement with the properties of the galactosyltransferase I activity measured in extracts of fibroblasts from a patient with galactosyltransferase I deficiency (14). The catalytically active allele, 4Gal-T7^186D, exhibited an approximately 2-fold lower K_m for the acceptor substrate MeUmb--Xyl, whereas the extract of patient cells had a 2-fold higher K_m for xylosyl-serine compared with control cells. Furthermore, 4Gal-T7^186D had a 4-fold higher K_m for the donor substrate, whereas extracts exhibited an almost 7-fold lower K_m than control cells. Additionally, only about 30-70% of the secreted decorin was devoid of the glycosaminoglycan chain, whereas the mature proteoglycan even contained longer dermatan sulfate chains. These discrepancies are likely related in part to experimental variation. Analysis of glycosyltransferase activities in extracts may be influenced by a number of factors. It is possible that multiple enzymes catalyze formation of the same linkage with different kinetic properties. Other factors in extracts may interfere or bind substrates. Furthermore, the properties of recombinant secreted forms of the enzyme may be different than those found with the transmembrane enzyme in cell homogenates. The total activity assessed in the patient fibroblasts was approximately 5% compared with controls, whereas both parents showed 50% reduction in activities. The 5% activity measured in the patient is likely to originate from the 4Gal-T7^186D allele. The recombinant form of this allele was comparatively more active (Table I), but its poorer kinetic properties, potentially combined with a lower stability in cells or extracts (14), may account for the reduction in activities observed in the patient and parents. One unexplained observation from the study of Quentin et al. (14) was that the 3-galactosyltransferase II activity forming the Gal1-3Gal1-4Xyl1-O-Ser structure was also reduced in the patient and parents. Further studies of the in vivo functions of the allelic variants of 4Gal-T7 are required to fully assess their functions in proteoglycan biosynthesis.

The identified mutations in 4Gal-T7 are in the putative catalytic domain and involve residues that are partly or fully conserved among members of the 4Gal-T gene family (Fig. 1A). The effects of these substitutions are in agreement with predictions based on x-ray crystallography data on the catalytic unit of 4Gal-T1.⁴ The region around Ala¹⁸⁶ corresponds to a segment in 4Gal-T1 that is included in the protein core. The adjacent Pro and His residues at positions 182 and 184 in 4Gal-T7 are strictly conserved among all human 4GalT sequences. Ala¹⁸⁶ of 4Gal-T7 corresponds to Ser²⁷⁴ in 4Gal-T1. Ser²⁷⁴ is in a region close (less than 5 angstrom distance) to the UDP-Gal binding site and may interact with this and hence explain the poorer kinetic properties of the 4Gal-T7^186D variant. The Gly²⁰¹-Gly²⁰² adjacent to Leu²⁰⁶ are strictly conserved among 4Gal-T sequences, and they are included in the catalytic pocket of 4Gal-T1. Leu²⁰⁶ is also strictly conserved in all sequences, and the corresponding Leu²⁹⁶ in 4Gal-T1 is included in a network of hydrophobic interactions in the protein core with aromatic residue Phe³⁰¹, Phe³⁰⁷, Phe²⁹⁰, and Leu³²⁵. The Pro²⁰⁶ substitution in 4Gal-T7^206P is predicted to markedly change the fold of the protein core, which is in agreement with the observed lack of activity of this variant.

Galactosyltransferase I has been considered as a cis-Golgi located enzyme in epiphyseal cartilage (32) and is believed to be noncovalently associated with the protein xylosyltransferase (33, 34). The sequence of 4Gal-T7 predicts relatively long cytoplasmic (28 residues) and transmembrane (30 residues) domains compared with other 4Gal-Ts and Golgi-located glycosyltransferases in general. The cytoplasmic domain and the stem region are hydrophilic. We have not identified putative motifs that are predicted to mediate binding to the protein xylosyltransferase. The availability of recombinant 4Gal-T7 and antibodies thereto may provide tools for studying the interaction and possibly cloning the xylosyltransferase.

Two homologues of the 4Gal-T gene family have been identified in the nematode Caenorhabtidis elegans (35). In a phylogenetic analysis presented by Lo et al. (36) the gene W02B12.11 (designated C. 2) (GenBank^TM accession number Z66521) clustered with the 4Gal-T5 and 4Gal-T6 subgroup, while the gene R10E11.4 (designated C. elegans 1) (GenBank^TM accession number Z29095) was not clustered. If 4Gal-T7 is included in this analysis, the R10E11.4 gene and 4Gal-T7 form a separate cluster (Fig. 1B). The close relationship between W02B12.11 and 4Gal-T1 to 4Gal-T6 is further supported by the finding that two of the four intron positions in W02B12.11 align with the conserved intron/exon boundaries in 4Gal-T1 to 4Gal-T4 (Fig. 1A). In addition, the predicted coding region of W02B12.11 includes the four conserved cysteine residues in 4Gal-T1 to 4Gal-T6. An evolutionary relationship between R10E11.4 and 4Gal-T7 is suggested by the sequence similarity, and this includes substitutions in the same positions in major conserved sequence motif among most 4Gal-Ts (Fig. 1). However, the substituted residues are not similar, and cysteine residues are not conserved, although two residues in the very C-terminal sequences do align between the two sequences (Fig. 1A). The coding region of R10E11.4 is organized in six exons, and none of the intron/exon boundaries align with those of 4Gal-T1 to 4Gal-T4, nor do they align with 4Gal-T7.

The R10E11.4 gene corresponds to the sqv-3 gene found to play a critical role in vulval invagination in C. elegans (37). Glycosyltransferase activity of the protein encoded by either of the C. elegans 4Gal-T homologues have not been reported to our knowledge, but recent expression of a soluble, secreted construct of R10E11.4 in insect cells demonstrated similar activity as reported here for human 4Gal-T7.⁵ Another gene found to play a role in vulval invagination in C. elegans is sqv-8 (37), which showed high sequence similarity to the recently cloned 1,3-glucuronosyltransferase that adds the fourth residue to the proteoglycan linkage region tetrasaccharide (GlcA1-3Gal1-3Gal1-4Xyl1-O-Ser) (38). The finding that impairment of the sqv-3 and sqv-8 genes in nematodes produce the same defect in vulval invagination would be in agreement with the hypothesis that both of these genes were involved in synthesis of the proteoglycan linkage region tetrasaccharide, albeit at different steps. Identification of the molecular genetic basis for the defect in proteoglycan biosynthesis of the patient studied here suggests that extensive studies of genetic defects in patients with progeroid syndromes and other inherited connective tissue disorders should be undertaken.

	ACKNOWLEDGEMENTS

We thank Dr. Louis Gastinel for sequence analysis of the 4Gal-T7 variants, Dr. Michael A. Hollingsworth for many helpful suggestions and critical reading of the manuscript, and Tom Caffrey and Naoaki Akisawa for technical assistance.

	FOOTNOTES

^* This work was supported by the Danish Cancer Society, the Velux Foundation, the Danish Research Council, Praxis Grant XXI 2/2.1/BIA/276/94, National Institutes of Health Resource Center for Biomedical Complex Carbohydrates Grant 5 P41 RR05351, and Deutsche Forschungsgemeinschaft Grant SFB 310, Project B2.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) AJ005382.

^** To whom correspondence should be addressed: School of Dentistry, Nørre Alle 20, DK-2200 Copenhagen N, Denmark. Tel.: 45-35326835; Fax: 45-35326505; E-mail: henrik.clausen@odont.ku.dk.

² H. Kresse, unpublished observation.

³ H. Kresse, unpublished observation.

⁴ Gastinel, L. N., Cambillau, C., and Bourne, Y. (1999) EMBO J. 18, 3546-3557.

⁵ R. Almeida and H. Clausen, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: 4Gal-T, UDP-galactose:-N-acetylglucosamine/-glucose/-xylose 1,4-galactosyltransferase; EST, expressed sequence tags; MeUmb, methylumbelliferyl; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum correlation; HMBC, heteronuclear multiple bond correlation; PCR, polymerase chain reaction.

	REFERENCES

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