Transmembrane Protein Topology Research Articles

A Genome Wide Screening Approach for Membrane-targeted Proteins

Membrane-associated proteins are critical for intra- and intercellular communication. Accordingly approaches are needed for rapid and comprehensive identification of all membrane-targeted gene products in a given cell or tissue. Here we describe a modification of the yeast Ras recruitment system to this end and designate the modified approach the Ras membrane trap (RMT). A pilot RMT screen was carried out on the central nervous system of the mollusk Lymnaea stagnalis, a model organism from a phylum that still lacks a representative with a sequenced genome. 112 gene products were identified in the screen of which 79 lack assignable homologs in available data bases. Currently available annotation tools predicted membrane association of only 45% of the 112 proteins, although experimental verification in mammalian cells confirmed membrane association for all clones tested. Thus, genome annotation using currently available tools is likely to underpredict representation of membrane-associated gene products. The 32 proteins with known homologies include many targeted to the endoplasmic reticulum or the nucleus, thus RMT provides a tool that can cover intracellular membrane proteomes. Two sequences were found to represent gene families not found to date in invertebrate genomes, emphasizing the need for whole genome sequences from mollusks and indeed from representatives of all major invertebrate phyla.

An improved hidden Markov model for transmembrane protein detection and topology prediction and its applications to complete genomes

Knowledge of the transmembrane helical topology can help identify binding sites and infer functions for membrane proteins. However, because membrane proteins are hard to solubilize and purify, only a very small amount of membrane proteins have structure and topology experimentally determined. This has motivated various computational methods for predicting the topology of membrane proteins. We present an improved hidden Markov model, TMMOD, for the identification and topology prediction of transmembrane proteins. Our model uses TMHMM as a prototype, but differs from TMHMM by the architecture of the submodels for loops on both sides of the membrane and also by the model training procedure. In cross-validation experiments using a set of 83 transmembrane proteins with known topology, TMMOD outperformed TMHMM and other existing methods, with an accuracy of 89% for both topology and locations. In another experiment using a separate set of 160 transmembrane proteins, TMMOD had 84% for topology and 89% for locations. When utilized for identifying transmembrane proteins from non-transmembrane proteins, particularly signal peptides, TMMOD has consistently fewer false positives than TMHMM does. Application of TMMOD to a collection of complete genomes shows that the number of predicted membrane proteins accounts for approximately 20-30% of all genes in those genomes, and that the topology where both the N- and C-termini are in the cytoplasm is dominant in these organisms except for Caenorhabditis elegans. http://liao.cis.udel.edu/website/servers/TMMOD/

Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database.

Functional genomic technologies are generating vast amounts of data describing the presence of transcripts or proteins in plant cells. Together with classical genetics, these approaches broaden our understanding of the gene products required for specific responses. Looking to the future, the focus of research must shift to the dynamic aspects of biology: molecular mechanisms of function and regulation. Phosphorylation is a key regulatory factor in all aspects of plant biology; but it is difficult, if not impossible, for most researchers to identify in vivo phosphorylation sites within their proteins of interest. We have developed a large-scale strategy for the isolation of phosphopeptides and identification by mass spectrometry (Nühse et al., 2003b). Here, we describe the identification of more than 300 phosphorylation sites from Arabidopsis thaliana plasma membrane proteins. These data will be a valuable resource for many fields of plant biology and overcome a major impediment to the elucidation of signal transduction pathways. We present an analysis of the characteristics of phosphorylation sites, their conservation among orthologs and paralogs, and the existence of putative motifs surrounding the sites. These analyses yield general principles for predicting other phosphorylation sites in plants and provide indications of specificity determinants for responsible kinases. In addition, more than 50 sites were mapped on receptor-like kinases and revealed an unexpected complexity of regulation. Finally, the data also provide empirical evidence on the topology of transmembrane proteins. This information indicates that prediction programs incorrectly identified the cytosolic portion of the protein in 25% of the transmembrane proteins found in this study. All data are deposited in a new searchable database for plant phosphorylation sites maintained by PlantsP (http://plantsp.sdsc.edu) that will be updated as the project expands to encompass additional tissues and organelles.

Best alpha-helical transmembrane protein topology predictions are achieved using hidden Markov models and evolutionary information.

Methods that predict the topology of helical membrane proteins are standard tools when analyzing any proteome. Therefore, it is important to improve the performance of such methods. Here we introduce a novel method, PRODIV-TMHMM, which is a profile-based hidden Markov model (HMM) that also incorporates the best features of earlier HMM methods. In our tests, PRODIV-TMHMM outperforms earlier methods both when evaluated on "low-resolution" topology data and on high-resolution 3D structures. The results presented here indicate that the topology could be correctly predicted for approximately two-thirds of all membrane proteins using PRODIV-TMHMM. The importance of evolutionary information for topology prediction is emphasized by the fact that compared with using single sequences, the performance of PRODIV-TMHMM (as well as two other methods) is increased by approximately 10 percentage units by the use of homologous sequences. On a more general level, we also show that HMM-based (or similar) methods perform superiorly to methods that focus mainly on identification of the membrane regions.

A Combined Transmembrane Topology and Signal Peptide Prediction Method

An inherent problem in transmembrane protein topology prediction and signal peptide prediction is the high similarity between the hydrophobic regions of a transmembrane helix and that of a signal peptide, leading to cross-reaction between the two types of predictions. To improve predictions further, it is therefore important to make a predictor that aims to discriminate between the two classes. In addition, topology information can be gained when successfully predicting a signal peptide leading a transmembrane protein since it dictates that the N terminus of the mature protein must be on the non-cytoplasmic side of the membrane. Here, we present Phobius, a combined transmembrane protein topology and signal peptide predictor. The predictor is based on a hidden Markov model (HMM) that models the different sequence regions of a signal peptide and the different regions of a transmembrane protein in a series of interconnected states. Training was done on a newly assembled and curated dataset. Compared to TMHMM and SignalP, errors coming from cross-prediction between transmembrane segments and signal peptides were reduced substantially by Phobius. False classifications of signal peptides were reduced from 26.1% to 3.9% and false classifications of transmembrane helices were reduced from 19.0% to 7.7%. Phobius was applied to the proteomes of Homo sapiens and Escherichia coli. Here we also noted a drastic reduction of false classifications compared to TMHMM/SignalP, suggesting that Phobius is well suited for whole-genome annotation of signal peptides and transmembrane regions. The method is available at http://phobius.cgb.ki.se/ as well as at http://phobius.binf.ku.dk/

Molecular Cloning of a Chondroitin Polymerizing Factor That Cooperates with Chondroitin Synthase for Chondroitin Polymerization

We recently cloned human chondroitin synthase (ChSy) exhibiting the glucuronyltransferase-II (GlcATII) and N-acetylgalactosaminyltransferase-II (GalNAcTII) activities responsible for the biosynthesis of repeating disaccharide units of chondroitin sulfate, but chondroitin polymerization was not demonstrated in vitro using the recombinant ChSy. We report here that the chondroitin polymerizing activity requires concomitant expression of a novel protein designated chondroitin polymerizing factor (ChPF) with ChSy. The human ChPF consists of 775 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 23% identity to that of human ChSy. The expression of a soluble recombinant form of the protein in COS-1 cells produced a protein with little GlcAT-II or GalNAcT-II activity. In contrast, coexpression of the ChPF and ChSy yielded markedly augmented glycosyltransferase activities, whereas simple mixing of the two separately expressed proteins did not. Moreover, using both UDP-glucuronic acid (GlcUA) and UDP-N-acetylgalactosamine (GalNAc) as sugar donors, chondroitin polymerization was demonstrated on the so-called glycosaminoglycan-protein linkage region tetrasaccharide sequence of alpha-thrombomodulin. These results suggested that the ChPF acts as a specific activating factor for ChSy in chondroitin polymerization. The coding region of the ChPF was divided into four discrete exons and localized to chromosome 2q35-q36. Northern blot analysis revealed that the ChPF gene exhibited a markedly different expression pattern among various human tissues, which was similar to that of ChSy. Thus, the ChPF is required for chondroitin polymerizing activity of mammalian ChSy.

Molecular Cloning and Expression of a Second Chondroitin N-Acetylgalactosaminyltransferase Involved in the Initiation and Elongation of Chondroitin/Dermatan Sulfate

We identified a novel human chondroitin N-acetylgalactosaminyltransferase, designated chondroitin GalNAcT-2 after a BLAST analysis of the GenBank(TM) data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GalNAcT-1. Like chondroitin GalNAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUA beta 1-3Gal beta 1-O-C(2)H(4)NHCbz, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GlcUA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GalNAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains.

The prediction of amphiphilic alpha-helices.

A number of sequence-based analyses have been developed to identify protein segments, which are able to form membrane interactive amphiphilic alpha-helices. Earlier techniques attempted to detect the characteristic periodicity in hydrophobic amino acid residues shown by these structure and included the Molecular Hydrophobic Potential (MHP), which represents the hydrophobicity of amino acid residues as lines of isopotential around the alpha-helix and analyses based on Fourier transforms. These latter analyses compare the periodicity of hydrophobic residues in a putative alpha-helical sequence with that of a test mathematical function to provide a measure of amphiphilicity using either the Amphipathic Index or the Hydrophobic Moment. More recently, the introduction of computational procedures based on techniques such as hydropathy analysis, homology modelling, multiple sequence alignments and neural networks has led to the prediction of transmembrane alpha-helices with accuracies of the order of 95% and transmembrane protein topology with accuracies greater than 75%. Statistical approaches to transmembrane protein modeling such as hidden Markov models have increased these prediction levels to an even higher level. Here, we review a number of these predictive techniques and consider problems associated with their use in the prediction of structure / function relationships, using alpha-helices from G-coupled protein receptors, penicillin binding proteins, apolipoproteins, peptide hormones, lytic peptides and tilted peptides as examples.

Molecular Cloning and Expression of Human ChondroitinN-Acetylgalactosaminyltransferase: THE KEY ENZYME FOR CHAIN INITIATION AND ELONGATION OF CHONDROITIN/DERMATAN SULFATE ON THE PROTEIN LINKAGE REGION TETRASACCHARIDE SHARED BY HEPARIN/HEPARAN SULFATE

Based on sequence homology with the recently cloned human chondroitin synthase, we identified a novel beta1,4-N-acetylgalactosaminyltransferase, which consisted of 532 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 27% identity to that of human chondroitin synthase. The expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc not only to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUAbeta1--3Galbeta1-O-C(2)H(4)NH-benzyloxycarbonyl, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein linkage region of chondroitin sulfate. Hence, the enzyme is involved in the biosynthetic initiation and elongation of chondroitin sulfate and is the key enzyme responsible for the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate or heparin/heparan sulfate chains. The coding region of this enzyme was divided into seven discrete exons and localized to chromosome 8. Northern blot analysis revealed that the chondroitin GalNAc transferase gene exhibited a ubiquitous but markedly differential expression in human tissues and that the expression pattern was similar to that of chondroitin synthase. Thus, more than two distinct enzymes forming the novel gene family are required for chain initiation and elongation in chondroitin/dermatan sulfate as in the biosynthesis of heparin/heparan sulfate.

Two closely related forms of UDP-GlcNAc: alpha6-D-mannoside beta1,2-N-acetylglucosaminyltransferase II occur in the clawed frog Xenopus laevis.

UDP-GlcNAc:alpha6-D-mannoside beta1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) is a medial-Golgi resident enzyme that catalyses an essential step in the biosynthetic pathway leading from high mannose to complex N-linked oligosaccharides. Screening a cDNA library from Xenopus laevis ovary with a human GnT II DNA probe resulted in the isolation of two cDNA clones encoding two closely related GnT II isoenzymes, GnT II-A and GnT II-B. Analysis of the corresponding genomic DNAs revealed that the open reading frame of both X. laevis GnT II genes resides within a single exon. The GnT II-A gene was found to be transcriptionally active in all X. laevis tissues tested. In contrast, expression of the GnT II-B gene was detected only in a limited number of tissues. Both GnT II-A and GnT II-B exhibit a type II transmembrane protein topology with a putative N-terminal cytoplasmic tail of 9 amino acids followed by a transmembrane domain of 18 residues, and a C-terminal luminal domain of 405 residues. The two proteins differ at 28 amino acid positions within their luminal regions. Heterologous expression of soluble forms of the enzymes in insect cells showed that GnT II-A and GnT II-B are both catalytically active and exhibit similar specific activities. Both recombinant proteins are modified with N-linked oligosaccharides. N-terminal deletion studies demonstrated that the first 49 amino acid residues are not essential for proper folding and enzymatic activity of X. laevis GnT II.

Molecular Cloning and Expression of a Human Chondroitin Synthase

We have identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with two keywords: "one transmembrane domain" and "galactosyltransferase family." The identified protein consists of 802 amino acids with a type II transmembrane protein topology. The protein showed weak homology to the beta1,3-galactosyltransferase family on the amino-terminal side and to the beta1,4-galactosyltransferase family on the carboxyl-terminal side. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active enzyme, which transferred not only the glucuronic acid (GlcUA) from UDP-[(14)C]GlcUA but also N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to the polymer chondroitin. Identification of the reaction products demonstrated that the enzyme was chondroitin synthase, with both beta1,3-GlcUA transferase and beta1,4-GalNAc transferase activities. The coding region of the chondroitin synthase was divided into three discrete exons and localized to chromosome 15. Northern blot analysis revealed that the chondroitin synthase gene exhibited ubiquitous but markedly differential expression in the human tissues examined. Thus, we demonstrated that analogous to human heparan sulfate polymerases, the single polypeptide chondroitin synthase possesses two glycosyltransferase activities required for chain polymerization.

The HMMTOP transmembrane topology prediction server.

The HMMTOP transmembrane topology prediction server predicts both the localization of helical transmembrane segments and the topology of transmembrane proteins. Recently, several improvements have been introduced to the original method. Now, the user is allowed to submit additional information about segment localization to enhance the prediction power. This option improves the prediction accuracy as well as helps the interpretation of experimental results, i.e. in epitope insertion experiments. HMMTOP 2.0 is freely available to non-commercial users at http://www.enzim.hu/hmmtop. Source code is also available upon request to academic users.

Tissues of the clawed frog Xenopus laevis contain two closely related forms of UDP-GlcNAc:alpha3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I.

UDP-GlcNAc:alpha3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101) is a medial-Golgi enzyme that is essential for the processing of oligomannose to hybrid and complex N-glycans. On the basis of highly conserved sequences obtained from previously cloned mammalian GnTI genes, cDNAs for two closely related GnTI isoenzymes were isolated from a Xenopus laevis ovary cDNA library. As typical for glycosyltransferases, both proteins exhibit a type II transmembrane protein topology with a short N-terminal cytoplasmic tail (4 amino acids); a transmembrane domain of 22 residues; a stem region with a length of 81 (isoenzyme A) and 77 (isoenzyme B) amino acids, respectively; and a catalytic domain consisting of 341 residues. The two proteins differ not only in length but also at 13 (stem) and 18 (catalytic domain) positions, respectively. The overall identity of the catalytic domains of the X. laevis GnTI isoenzymes with their mammalian and plant orthologues ranges from 30% (Nicotiana tabacum) to 67% (humans). Isoenzymes A and B are encoded by two separate genes that were both found to be expressed in all tissues examined, albeit in varying amounts and ratios. On expression of the cDNAs in the baculovirus/insect cell system, both isoenzymes were found to exhibit enzymatic activity. Isoenzyme B is less efficiently folded in vivo and thus appears of lower physiological relevance than isoenzyme A. However, substitution of threonine at position 223 with alanine was sufficient to confer isoenzyme B with properties similar to those observed for isoenzyme A.

A novel tool for the prediction of transmembrane protein topology based on a statistical analysis of the SwissProt database: the OrienTM algorithm.

OrienTM is a computer software that utilizes an initial definition of transmembrane segments to predict the topology of transmembrane proteins from their sequence. It uses position-specific statistical information for amino acid residues which belong to putative non-transmembrane segments derived from statistical analysis of non-transmembrane regions of membrane proteins stored in the SwissProt database. Its accuracy compares well with that of other popular existing methods. A web-based version of OrienTM is publicly available at the address http://biophysics.biol.uoa.gr/OrienTM.

Predicting transmembrane protein topology with a hidden markov model: application to complete genomes

We describe and validate a new membrane protein topology prediction method, TMHMM, based on a hidden Markov model. We present a detailed analysis of TMHMM’s performance, and show that it correctly predicts 97–98 % of the transmembrane helices. Additionally, TMHMM can discriminate between soluble and membrane proteins with both specificity and sensitivity better than 99 %, although the accuracy drops when signal peptides are present. This high degree of accuracy allowed us to predict reliably integral membrane proteins in a large collection of genomes. Based on these predictions, we estimate that 20–30 % of all genes in most genomes encode membrane proteins, which is in agreement with previous estimates. We further discovered that proteins with Nin-Cin topologies are strongly preferred in all examined organisms, except Caenorhabditis elegans, where the large number of 7TM receptors increases the counts for Nout-Cin topologies. We discuss the possible relevance of this finding for our understanding of membrane protein assembly mechanisms. A TMHMM prediction service is available at http://www.cbs.dtu.dk/services/TMHMM/.

Molecular Cloning and Expression of a Novel Chondroitin 6-O-Sulfotransferase

A novel human chondroitin 6-O-sulfotransferase, designated C6ST-2, was identified by BLAST analysis of expressed sequence tag using the sequence of a previously described human chondroitin 6-O-sulfotransferase (C6ST-1) as a probe. The new cDNA sequence revealed an open reading frame coding for a protein of 486 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 24% identity to the human C6ST-1, and the highest sequence identity was found in the COOH-terminal catalytic domain. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active sulfotransferase with marked specificity for polymer chondroitin. In contrast, keratan sulfate and oligosaccharides containing the Galbeta1-4GlcNAc sequence, which are good acceptor substrates for the C6ST-1, hardly served as acceptors. The identification of the reaction product indicated that the enzyme is a novel chondroitin 6-O-sulfotransferase (C6ST-2) that mainly transfers sulfate to N-acetylgalactosamine. The coding region of C6ST-2 was contained in a single exon and localized to chromosome Xp11. Northern blot analysis of human brain poly(A)(+) RNA revealed a single transcript of 2.4 kilobase pairs. Reverse transcription-polymerase chain reaction analysis showed that C6ST-2 is developmentally regulated in various tissues with expression persisting through adulthood in the spleen. Thus, we demonstrated the redundancy in chondroitin 6-O-sulfotransferases capable of forming chondroitin 6-sulfate, which is important for understanding the mechanisms leading to specific changes in the sulfation profile of chondroitin sulfate chains in various tissues during development and malignant transformation.

Molecular cloning and functional expression of β1,2-xylosyltransferase cDNA from Arabidopsis thaliana

The transfer of xylose from UDP-xylose to the core β-linked mannose of N-linked oligosaccharides by β1,2-xylosyltransferase (XylT) is a widespread feature of plant glycoproteins which renders them immunogenic and allergenic in man. Here, we report the isolation of the Arabidopsis thaliana XylT gene, which contains two introns and encodes a 60.2 kDa protein with a predicted type II transmembrane protein topology typical for Golgi glycosyltransferases. Upon expression of A. thaliana XylT cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited XylT activity in vitro. Furthermore, the recombinant enzyme displayed XylT activity in vivo in the insect cells, as judged by the acquired cross-reaction of cellular glycoproteins with antibodies against the β1,2-xylose epitope. The cloned XylT cDNA as well as the recombinant enzyme are essential tools to study the role of β1,2-xylose in the immunogenicity and allergenicity of plant glycoproteins at the molecular level.

Cloning and Expression of a Novel Galactoside β1,3-Glucuronyltransferase Involved in the Biosynthesis of HNK-1 Epitope

We isolated a cDNA encoding a novel glucuronyltransferase, designated GlcAT-D, involved in the biosynthesis of the HNK-1 carbohydrate epitope from rat embryo cDNA by the degenerate polymerase chain reaction method. The new cDNA sequence revealed an open reading frame coding for a protein of 324 amino acids with type II transmembrane protein topology. The amino acid sequence of GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of GlcAT-D in COS-7 cells resulted in the formation of the HNK-1 epitope on the cell surface. The enzyme expressed in COS-7 cells transferred a glucuronic acid (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N-acetyllactosamine structure, but also to paragloboside (lacto-N-neotetraosylceramide), a precursor of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity analysis using a soluble chimeric form of GlcAT-D revealed that GlcAT-D transfers a GlcA not only to Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ + but also to Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ +. Enzymatic hydrolysis and Smith degradation of the reaction product indicated that GlcAT-D transfers a GlcA through a beta1,3-linkage to a terminal galactose. The GlcAT-D transcripts were detected in embryonic, postnatal, and adult rat brain. In situ hybridization analysis revealed that the expression pattern of GlcAT-D transcript in embryo is similar to that of GlcAT-P, but distinct expression of GlcAT-D was observed in the embryonic pallidum and retina. Regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive, indicating that both GlcATs are involved in the synthesis of the HNK-1 epitope in vivo.

2PA060 膜貫通タンパク質データベースの構築と膜貫通トポロジー解析

2PA060 膜貫通タンパク質データベースの構築と膜貫通トポロジー解析

Molecular Cloning and Expression of Glucuronyltransferase I Involved in the Biosynthesis of the Glycosaminoglycan-Protein Linkage Region of Proteoglycans

We isolated a cDNA encoding a novel glucuronyltransferase from human placenta cDNA with the use of the degenerate reverse transcriptase-polymerase chain reaction method. Degenerate primers were designed based upon the amino acid sequence alignment of rat glucuronyltransferase (GlcAT-P) involved in the biosynthesis of the carbohydrate epitope HNK-1 with putative proteins in Caenorhabditis elegans and Schistosoma mansoni. The new cDNA sequence revealed an open reading frame coding for a protein of 335 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 43% identity to the rat GlcAT-P, and the highest sequence identity was found in the COOH-terminal catalytic domain. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active glucuronyltransferase with marked specificity for a glycoserine Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. In contrast, asialoorosomucoid, which contains the Galbeta1-4GlcNAc sequence and is a good acceptor substrate for the GlcAT-P, did not serve as an acceptor. The reaction product was sensitive to beta-glucuronidase digestion and co-chromatographed with authentic GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser in high-performance liquid chromatography, suggesting that the enzyme is a beta1, 3-glucuronyltransferase. These results indicate that this new member of the glucuronyltransferase gene family is the enzyme previously described as glucuronyltransferase I that forms the glycosaminoglycan-protein linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans.