Large-scale mutational analysis in the EXT1 and EXT2 genes for Japanese patients with multiple osteochondromas.

BACKGROUND: Multiple osteochondroma (MO) is an autosomal dominant skeletal disorder characterized by the formation of multiple osteochondromas, and exostosin-1 (EXT1) and exostosin-2 (EXT2) are major causative genes in MO. In this study, we evaluated the genetic backgrounds and mutational patterns in Japanese families with MO. RESULTS: We evaluated 112 patients in 71 families with MO. Genomic DNA was isolated from peripheral blood leucocytes. The exons and exon/intron junctions of EXT1 and EXT2 were directly sequenced after PCR amplification. Fifty-two mutations in 47 families with MO in either EXT1 or EXT2, and 42.3% (22/52) of mutations were novel mutations. Twenty-nine families (40.8%) had mutations in EXT1, and 15 families (21.1%) had mutations in EXT2. Interestingly, three families (4.2%) had mutations in both EXT1 and EXT2. Twenty-four families (33.8%) did not exhibit mutations in either EXT1 or EXT2. With regard to the types of mutations identified, 59.6% of mutations were inactivating mutations, and 38.5% of mutations were missense mutations. CONCLUSIONS: We found that the prevalence of EXT1 mutations was greater than that of EXT2 mutations in Japanese MO families. Additionally, we identified 22 novel EXT1 and EXT2 mutations in this Japanese MO cohort. This study represents the variety of genotype in MO.

Wisteria floribunda agglutinin positive human Mac-2-binding protein as a predictor of hepatocellular carcinoma development in chronic hepatitis C patients.

AIMS: Wisteria floribunda agglutinin (WFA)-positive human Mac-2-binding protein (WFA(+) -M2BP) is a new glycol marker related to liver fibrosis. The aim of the present study was to evaluate WFA(+) -M2BP as a predictor of hepatocellular carcinoma (HCC) development in patients with chronic hepatitis C. METHODS: This case-control study included 14 patients with chronic hepatitis C who developed HCC and 52controls, matched for age, gender, and fibrosis stage. WFA(+) -M2BP was measured at biopsy and follow-up. Time zero was set at the date of liver biopsy. RESULTS: WFA(+) -M2BP increased stepwise with progression of liver fibrosis (p < 0.001). Cumulative incidence of HCC development was significantly higher in patients with WFA(+) -M2BP ≥4.2 (p < 0.001) or in those with time-course changes in WFA(+) -M2BP (ΔWFA(+) -M2BP/year) ≥0.3 (p = 0.03). Multivariate analyses demonstrated that WFA(+) -M2BP ≥4.2 [hazard ratio (HR): 4.1, 95% confidence interval (CI): 1.1-15, p = 0.04], ΔWFA(+) -M2BP/year ≥0.3 (HR: 5.5, 95% CI: 1.5-19, p = 0.008), and AFP ≥10 ng/ml (HR: 4.7, 95% CI: 1.1-19, p = 0.03) were independent predictive factors of HCC development. Based on these data, we developed a simple scoring system to predict HCC development using these three factors. Using these scores, patients were classified into four groups; cumulative incidence of HCC development significantly increased with increasing scores (p < 0.001). CONCLUSIONS: WFA(+) -M2BP measurements and time-course changes in WFA(+) -M2BP can be used to identify patients at high risk of HCC development. Real-time monitoring of WFA(+) -M2BP can be a novel predictor of HCC development.

Application of a glycoproteomics-based biomarker development method: alteration in glycan structure on colony stimulating factor 1 receptor as a possible glycobiomarker candidate for evaluation of liver cirrhosis.

The importance of diagnosis and therapies for liver cirrhosis (LC) is indisputable. Thus, a reliable method for monitoring the progression of liver fibrosis and resultant LC is urgently needed. Previously, using a lectin-assisted glycoproteomic method, we identified 26 serum glycoproteins as promising glycobiomarker candidates for monitoring the progression of liver diseases. In this study, we identified colony stimulating factor 1 receptor (CSF1R) as a promising LC marker candidate and then established Wisteria floribunda agglutinin (WFA)-reactive CSF1R (WFA(+)-CSF1R) as a novel possible glycobiomarker candidate by utilizing a glycoproteomics-based strategy. The serum level of WFA(+)-CSF1R in patients with hepatitis C virus (HCV)-infected liver disease was measured by an antibody-lectin sandwich ELISA. In a proof-of-concept experiment of the strategy preceding to future clinical studies, LC patients showed a high serum WFA(+)-CSF1R level in selected samples (P = 1.3 × 10(-17)). This result suggests WFA(+)-CSF1R is a possible biomarker candidate for evaluation of LC. Our results verified feasibility of this strategy for glycobiomarker development.

Novel glycobiomarker for ovarian cancer that detects clear cell carcinoma.

Epithelial ovarian cancer (EOC) is often asymptomatic and thus diagnosed at advanced stages with a poor prognosis. False-negative results for the conventional marker CA125 frequently occur in cases of clear cell carcinoma (CCC), a type of EOC; therefore, it is necessary to develop biomarkers with greater sensitivity. We previously reported a strategy to discover glycobiomarker candidates by combined lectin microarray and IGOT-LC/MS analysis. We have now optimized this strategy for discovering EOC biomarkers. Glycopeptides possessing cancerous glycans were enriched from the ascites fluids and culture supernatants of cancer cell lines with a fucose-binding lectin, AAL. IGOT-LC/MS analysis of CCC samples yielded 144 candidate glycoproteins. We selected WFA by lectin microarray as the optimal lectin to distinguish EOC from gastric and colon cancer. The candidates were narrowed by Western analysis of the WFA-bound fraction of ascites fluids. One of the final candidates, WFA-reactive ceruloplasmin, produced higher signals in the ascites fluids of EOC patients, including CCC, in comparison with the benign samples, while CA125 levels were comparable in the sandwich ELISA. Thus, our glycoproteomic strategy featuring efficient enrichment of glycans with disease-related alterations is applicable to various diseases.

Chondroitin sulfate synthase-2/chondroitin polymerizing factor has two variants with distinct function.

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.

Involvement of AP-2rep in morphogenesis of the axial mesoderm in Xenopus embryo.

We have previously isolated a cDNA clone coding for Xenopus AP-2rep (activator protein-2 repressor), a member of the Krüppel-like factor family, and reported its expression pattern in developing Xenopus embryos. In the present study, the physiological function of AP-2rep in the morphogenetic movements of the dorsal mesoderm and ectoderm was investigated. Embryos injected with either AP-2rep or VP16repC (a dominant-negative mutant) into the dorsal marginal zone at the 4-cell stage exhibited abnormal morphology in dorsal structures. Both AP-2rep and VP16repC also inhibited the elongation of animal cap explants treated with activin without affecting the expression of differentiation markers. Whole-mount in situ hybridization analysis revealed that expression of brachyury and Wnt11 was greatly suppressed by injection of VP16repC or AP-2rep morpholino, but expression was restored by the simultaneous injection of wild-type AP-2rep RNA. Furthermore, the morphogenetic abnormality induced by injection of VP16repC or AP-2rep morpholino was restored by simultaneous injection of brachyury or Wnt11 mRNA. These results show that AP-2rep is involved in the morphogenesis of the mesoderm at the gastrula stage, via the brachyury and/or Wnt pathways.

A novel beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T8), which synthesizes poly-N-acetyllactosamine, is dramatically upregulated in colon cancer.

A new member of the UDP-N-acetylglucosamine: beta-galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) family having the beta3-glycosyltransferase motifs was identified using an in silico method. This novel beta3Gn-T was cloned from a human colon cancer cell line and named beta3Gn-T8 based on its position in a phylogenetic tree and enzymatic activity. Beta3Gn-T8 transfers GlcNAc to the non-reducing terminus of the Galbeta1-4GlcNAc of tetraantennary N-glycan in vitro. HCT15 cells transfected with beta3Gn-T8 cDNA showed an increase in reactivity to both LEA and PHA-L4 in a flow cytometric analysis. These results indicated that beta3Gn-T8 is involved in the biosynthesis of poly-N-acetyllactosamine chains on tetraantennary (beta1,6-branched) N-glycan. In most of the colorectal cancer tissues examined, the level of beta3Gn-T8 transcript was significantly higher than in normal tissue. Beta3Gn-T8 could be an enzyme involved in the synthesis of poly-N-acetyllactosamine on beta1-6 branched N-glycans in colon cancer.

Clinicopathological characteristics and diagnostic performance of Wisteria floribunda agglutinin positive Mac-2-binding protein as a preoperative serum marker of liver fibrosis in hepatocellular carcinoma.

BACKGROUND: Wisteria floribunda agglutinin positive Mac-2-binding protein (WFA(+)-M2BP) is a novel serum marker of liver fibrosis identified in glycoproteomic biomarker screening studies, and its clinicopathological characteristics have yet to be elucidated sufficiently for clinical utilization. METHODS: We retrospectively analyzed the clinicopathology data and serum WFA(+)-M2BP levels in 376 hepatocellular carcinoma patients undergoing liver surgery. WFA(+)-M2BP was quantified in frozen serum samples collected at the time of surgery using the FastLec-Hepa method. RESULTS: Significant independent determinants of serum WFA(+)-M2BP levels included pathological diagnosis of cirrhosis, female gender, hepatitis C virus (HCV) infection, and liver dysfunction characteristics, such as abnormal indocyanine green retention rate at 15 min, platelet counts, albumin levels, alanine aminotransferase levels, and total bilirubin levels. Serum WFA(+)-M2BP levels increased with the pathological fibrosis stage and liver dysfunction severity. HCV infection significantly affected serum WFA(+)-M2BP levels throughout the pathological and functional progression of liver fibrosis, and the effect of gender was significant only in F4 stage patients with severe liver dysfunction. The diagnostic thresholds for cutoff index values for cirrhosis were 1.435 and 4.615 in HCV-negative and HCV-positive patients, respectively. Serum WFA(+)-M2BP levels at the time of operation were a significant predictor of hepatocellular carcinoma recurrence and overall survival in both HCV-negative and HCV-positive patients. CONCLUSIONS: Serum WFA(+)-M2BP levels reflected both the pathological and functional progression of liver fibrosis comprehensively and continuously. Elevated WFA(+)-M2BP levels were a significant risk factor for tumor recurrence and decreased overall survival after liver surgery independent of HCV infection.

A novel serum marker, glycosylated Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA(+)-M2BP), for assessing liver fibrosis.

BACKGROUND: Recently, a novel marker, hyperglycosylated Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA(+)-M2BP), was developed for liver fibrosis using the glycan "sugar chain"-based immunoassay; however, the feasibility of WFA(+)-M2BP for assessing liver fibrosis has not been proven with clinical samples of hepatitis. METHODS: Serum WFA(+)-M2BP values were evaluated in 200 patients with chronic liver disease who underwent histological examination of liver fibrosis. The diagnostic accuracy of WFA(+)-M2BP values was compared with various fibrosis markers, such as ultrasound based-virtual touch tissue quantification (VTTQ), magnetic resonance imaging based-liver-to-major psoas muscle intensity ratio (LMR), and serum markers, including hyaluronic acid, type 4 collagen, and aspartate transaminase to platelet ratio index (APRI). RESULTS: Serum WFA(+)-M2BP levels in patients with fibrosis grades F0, F1, F2, F3, and F4 had cutoff indices 1.62, 1.82, 3.02, 3.32, and 3.67, respectively, and there were significant differences between fibrosis stages F1 and F2, and between F2 and F3 (P < 0.01). The area under the receiver operating characteristic curves for the diagnosis of fibrosis (F ≥ 3) using serum WFA(+)-M2BP values (0.812) was almost comparable to that using VTTQ examination (0.814), but was superior to the other surrogate markers, including LMR index (0.766), APRI (0.694), hyaluronic acid (0.683), and type 4 collagen (0.625) (P < 0.01 each). CONCLUSIONS: Serum WFA(+)-M2BP values based on a glycan-based immunoassay is an accurate, reliable, and reproducible method for the assessment of liver fibrosis. This approach could be clinically feasible for evaluation of beneficial therapy through the quantification of liver fibrosis in hepatitis patients if this measurement application is commercially realized.

Molecular cloning and characterization of beta1,4-N-acetylgalactosaminyltransferases IV synthesizing N,N'-diacetyllactosediamine.

A sequence highly homologous to beta1,4-N-acetylgalactosaminyltransferase III (beta4GalNAc-T3) was found in a database of human expressed sequence tags. The full-length open reading frame of the gene, beta4GalNAc-T4 (GenBank accession number AB089939), was cloned using the 5' rapid amplification of cDNA ends method. It encodes a typical type II transmembrane protein of 1039 amino acids having 42.6% identity with beta4GalNAc-T3. The recombinant enzyme transferred N-acetylgalactosamine to N-acetylglucosamine-beta-benzyl with a beta1,4-linkage to form N,N'-diacetyllactosediamine as did beta4GalNAc-T3. In specificity toward oligosaccharide acceptor substrates, it was quite similar to beta4GalNAc-T3 in vitro, however, the tissue distributions of the two enzymes were quite different. These results indicated that the two enzymes have similar roles in different tissues.

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 plays a critical role in chondroitin sulfate synthesis in cartilage.

Cartilage destruction leads to severe joint diseases, such as osteoarthritis and spinal disorders with back pain, and cartilage regeneration is very inefficient. A major component of the cartilage extracellular matrix is the proteoglycan aggrecan that contains approximately 100 chondroitin sulfate (CS) chains, which impart water absorption and resistance to compression. Here, we demonstrate that chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) plays a critical role in CS biosynthesis in cartilage. By in situ hybridization and real time reverse transcription-PCR of developing cartilage, CSGalNAcT-1 exhibited the highest level of expression. Its expression in chondrogenic ATDC5 cells correlated well with that of aggrecan core protein. In heterozygote and homozygote aggrecan-null cartilage where aggrecan transcription is decreased, CSGalNAcT-1 transcription diminished accordingly. Overexpression of the enzyme in chondrocytic cells further enhanced CS biosynthesis but not that of the aggrecan core protein, indicating that the enzyme activity is not saturated in the cells and that aggrecan synthesized in the overexpressing cells is heavier than the native molecule. Analysis of the CS chains synthesized in the overexpressing cells by gel chromatography and that of disaccharide composition revealed that the CS chains had similar length and sulfation patterns. Furthermore, adenoviral gene delivery of the enzyme into intervertebral discs displayed a substantial increase in the level of CS biosynthesis. These observations indicate that CSGalNAcT-1 overexpression increases the number of CS chains attached to aggrecan core protein. Our studies may lead to a new therapeutic intervention, ameliorating the outcome of cartilage degenerative diseases.

Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain.

Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.

Apical Golgi localization of N,N'-diacetyllactosediamine synthase, beta4GalNAc-T3, is responsible for LacdiNAc expression on gastric mucosa.

beta1,4-N-acetylgalactosaminyltransferase III (beta4GalNAc-T3), which was recently cloned and identified, exhibits GalNAc transferase activity toward a GlcNAcbeta residue with beta1,4-linkage, forming the N,N'-diacetyllactosediamine, GalNAcbeta1,4GlcNAc (LacdiNAc or LDN). Though LacdiNAc has not been found in the gastric mucosa, a large amount of transcript was detected in our previous study. To increase our knowledge of beta4GalNAc-T3 expression and its product LacdiNAc, we examined the exact localization of beta4GalNAc-T3 in human gastric mucosa using a newly developed antibody, monoclonal antibody (mAb) K1356. This antibody specifically detected the enzyme that transfected the beta4GalNAc-T3 gene into MKN45 cells, and the terminal betaGalNAc epitope yielded on the cell surface was recognized by a lectin, Wisteria floribunda agglutinin (WFA). beta4GalNAc-T3 was localized in the supra-nuclear region of surface mucous cells in gastric mucosa, and WFA positively stained the mucins secreted by the cells. In contrast, in the cells of the glandular compartment in the fundic glands and a few cells in the pyloric glands, beta4GalNAc-T3 was observed in the basolateral position of the nucleus, where no WFA reactivity was detected. The anti-Tn (GalNAcalpha-O-Ser/Thr) antibody staining did not overlap with the WFA staining. By measuring the binding activity of WFA using automated frontal affinity chromatography (FAC), we found WFA to bind most strongly LacdiNAc among the sugar chains examined. Neither beta4GalNAc-T3 nor WFA-positive staining was detected in intestinal metaplastic cells. These results suggest that the supra-nuclear expression of beta4GalNAc-T3 is essential for the formation of LacdiNAc on the surface mucous cells and that LacdiNAc and beta4GalNAc-T3 are novel differentiation markers of surface mucous cells in the gastric mucosa.

Complementary expression of AP-2 and AP-2rep in ectodermal derivatives of Xenopus embryos.

In an attempt to define the pattern of developmental expression of AP-2rep and AP-2 in Xenopus embryos, we cloned a Xenopus AP-2rep cDNA. The AP-2rep message was localized in the organizer region at the gastrula stage whereas AP-2 was expressed ventro-laterally in the animal hemisphere. Later, AP-2rep was expressed in the entire neural tissue at the neurula stage while AP-2 was predominantly expressed in the cranial neural crest areas. The endogenous expression of AP-2 in the neural crest area was diminished by ectopic injection of AP-2rep RNA, suggesting a role for AP-2rep in the differentiation of neural tissues by restricting the expression of AP-2 in the Xenopus embryo.

Comparison of glycosyltransferase families using the profile hidden Markov model.

In order to investigate the relationship between glycosyltransferase families and the motif for them, we classified 47 glycosyltransferase families in the CAZy database into four superfamilies, GTS-A, -B, -C, and -D, using a profile Hidden Markov Model method. On the basis of the classification and the similarity between GTS-A and nucleotidylyltransferase family catalyzing the synthesis of nucleotide-sugar, we proposed that ancient oligosaccharide might have been synthesized by the origin of GTS-B whereas the origin of GTS-A might be the gene encoding for synthesis of nucleotide-sugar as the donor and have evolved to glycosyltransferases to catalyze the synthesis of divergent carbohydrates. We also suggested that the divergent evolution of each superfamily in the corresponding subcellular component has increased the complexities of eukaryotic carbohydrate structure.

A BMP-4-dependent transcriptional control element in the 5' flanking region of Xenopus SCL gene.

We isolated 5.5kb genomic DNA fragment of Xenopus stem cell leukemia (SCL) that contains approximately 1.5kb of the 5' flanking region and 4.0kb of the first intron between a non-coding exon (exon 1) and a coding exon (exon 2). Sequencing result of the 5' flanking region has shown that there is a portion that shares 85% and 69% with the sequences of avian and mammalian genomes of SCL promoter region (-64 to +73). The 1.5kb 5' flanking region of SCL genome and various deletion constructs were inserted at the upstream of luciferase (luc) gene and used for the reporter assay. The reporter activity was first detected at the neurula stage in the embryos injected with -167+157/luc at the 2-cell stage and the values increased as the stages advanced. The experiments using dominant-negative constructs revealed that the activation of SCL transcription via the 5' flanking region requires the BMP-4 and GATA factors. Taken together with the in situ hybridization analysis indicating that expression of SCL was downregulated in the central nervous system in BMP-depleted embryos, the proximal sequence of SCL consists of a stage-dependent and BMP signaling-dependent control element.

A versatile planar QCM-based sensor design for nonlabeling biomolecule detection.

Despite high theoretical sensitivity, low-cost manufacture, and compactness potentially amenable to lab-on-a-chip use, practical hurdles have stymied the application of the quartz crystal microbalance (QCM) for aqueous applications such as detection of biomolecular interactions. The chief difficulty lies in achieving a sufficiently stable resonance signal in the presence of even minute fluctuations in hydrostatic pressure. In this work, we present a novel versatile planar sensor chip design (QCM chip) for a microliter-scale on-line biosensor. By sealing the quartz resonator along its edges to a flat, solid support, we provide uniform support for the crystal face not exposed to solvent, greatly decreasing deformation of the crystal resonator under hydrostatic pressure. Furthermore, this cassette design obviates the need for direct handling when exchanging the delicate quartz crystal in the flow cell. A prototype 27-MHz sensor signal exhibited very low noise over a range of flow rates up to 100 microL/min. In contrast, signals obtained from a conventional QCM sensor employing an O-ring-based holder were less stable and deteriorated even further with increasing flow rate. Additional control designs with intermediate amounts of unsupported undersurface yielded intermediate levels of stability, consistent with the interpretation that deformation of the crystal resonator under fluctuating hydraulic pressure is the chief source of noise. As a practical demonstration of the design's high effective sensitivity, we readily detected interaction between myoglobin and surface-bound antibody.

The high specificities of Phaseolus vulgaris erythro- and leukoagglutinating lectins for bisecting GlcNAc or beta 1-6-linked branch structures, respectively, are attributable to loop B.

Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E(4)- and L(4)-PHA) are unique in recognizing larger structures. E(4)- and L(4)-PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a beta1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E(4)- and L(4)-PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E(4)- and L(4)-PHA toward bisecting GlcNAc and beta1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E(4)- and L(4)-PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a beta1,6-linked branch.

Initiation of O-glycan synthesis in IgA1 hinge region is determined by a single enzyme, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2.

The hinge region of human immunoglobulin A1 (*IgA1) possesses multiple O-glycans, of which synthesis is initiated by the addition of GalNAc to serine or threonine through the activity of UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). We found that six pp-GalNAc-Ts, pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9, were expressed in B cells, IgA-bearing B cells, and NCI-H929 IgA myeloma cells. pp-GalNAc-T activities of these six enzymes for a synthetic IgA hinge peptide, which has nine possible O-glycosylation sites, were examined using a reversed phase-high performance liquid chromatography, a matrix-assisted laser desorption ionization time of flight mass spectrometry, and peptide sequencing analysis. pp-GalNAc-T2 showed the strongest activity transferring GalNAc to a maximum of eight positions. Other pp-GalNAc-Ts exhibited different substrate specificities from pp-GalNAc-T2; however, their activities were extremely weak. It was reported that the IgA1 hinge region possesses a maximum of five O-glycans, and their amino acid positions have been determined. We found that pp-GalNAc-T2 selectively transferred GalNAc residues to the same five positions. These results strongly suggested that pp-GalNAc-T2 is an essential enzyme for initiation of O-linked glycosylation of the IgA1 hinge region.

Chondroitin sulfate synthase-3. Molecular cloning and characterization.

Recently, it has become evident that chondroitin sulfate (CS) glycosyltransferases, which transfer glucuronic acid and/or N-acetylgalactosamine residues from each UDP-sugar to the nonreducing terminus of the CS chain, form a gene family. We report here a novel human gene (GenBank trade mark accession number AB086062) that possesses a sequence homologous with the human chondroitin sulfate synthase-1 (CSS1) gene, formerly known as chondroitin synthase. The full-length open reading frame consists of 882 amino acids and encodes a typical type II membrane protein. This enzyme contains a beta 3-glycosyltransferase motif and a beta 4-glycosyltransferase motif similar to that found in CSS1. Both the enzymes were expressed in COS-7 cells as soluble proteins, and their enzymatic natures were characterized. Both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, CS polymer, and their corresponding oligosaccharides were used as the acceptor substrates, but no polymerization reaction was observed as in the case of CSS1. The new enzyme was thus designated chondroitin sulfate synthase-3 (CSS3). However, the specific activity of CSS3 was much lower than that of CSS1. The reaction products were shown to have a GlcUA beta 1-3GalNAc linkage and a GalNAc beta 1-4GlcUA linkage in the nonreducing terminus of chondroitin resulting from glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcript level of CSS3 was much lower than that of CSS1, although it was ubiquitously expressed in various human tissues. These results indicate that CSS3 is a glycosyltransferase having both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. It may make a contribution to CS biosynthesis that differs from that of CSS1.