Does GD2 synthase (GD2S) detect cancer stem cells in blood samples of breast carcinomas?

INTRODUCTION: Cancer stem cells (CSCs) are a theorized subset of cells within the tumor that is thought to drive disease recurrence and metastatic spread. The aim of this study is to investigate mRNA and protein levels of ganglioside GD2 synthase (GD2S), in breast cancer (BC) patients. METHODS: 65 PBMCs of preoperative BC patients without chemotherapy were compared to PBMCs after chemotherapy and controls. RESULTS: GD2S were significantly higher in BC patients after chemotherapy compared to pre-chemotherapy at both mRNA and protein. GD2S was higher in pre-chemotherapy blood samples compared to control samples. CONCLUSIONS: Higher expression of GD2S in BC samples compared to healthy control indicates the potential utility of GD2S as a marker of malignancy.

Multidisciplinary management of anti-PP1Pk or anti-P alloimmunization during pregnancy: A new case with anti-P and a literature review.

BACKGROUND: Red blood cell alloimmunization is the first cause of fetal and neonatal anemia. Alloimmunizations with anti-PP1Pk or anti-P can cause recurrent miscarriages and hemolytic disease of the fetus and newborn in the 2nd and 3rd trimesters of pregnancy. We report on a pregnant patient immunized with anti-P and a history of recurrent miscarriages. CASE REPORT: This P2k (GLOB:-1; P1PK:-1,3) patient had a first pregnancy marked by a caesarean at 38 weeks of gestation (WG) for non-reassuring fetal heart rate. Then, she had three early spontaneous miscarriages. The fifth pregnancy began with a high titer of anti-P at 128. Early initiation of treatment with Intravenous Immunoglobulins (IVIg) and plasma exchanges (PE) starting at 5 WG permitted us to reduce the titer of anti-P below 32. A healthy infant was delivered by caesarean at 38 WG without anemia at birth and no exchange transfusion was required. DISCUSSION AND REVIEW OF THE LITERATURE: The P and Pk antigens are expressed on placental, trophoblastic, and embryonic cells. This explains why P1k (GLOB:-1; P1PK:1,3), P2k (GLOB:-1; P1PK:-1,3), or Tj(a-)/p (GLOB:-1; P1PK:-1,-3) patients are prone to recurrent abortions in the first trimester of pregnancy. A literature review demonstrated 87% (68/78) of miscarriages in p patients. However, publication biases are possible with the most severe cases being reported. CONCLUSION: Immunizations to P and PP1Pk antigens differ from others in their physiopathology and precocity. The association of PE and IVIg seems to be an effective treatment in the management of anti-PP1Pk or anti-P fetomaternal incompatibilities.

TLR7 in B cells promotes renal inflammation and Gd-IgA1 synthesis in IgA nephropathy.

TLR7 has been linked to the pathogenesis of glomerulonephritis, but its precise roles are not clear. In this study, we evaluated the roles of TLR7 in IgA nephropathy (IgAN). TLR7 proteins were abundant in CD19+ B cells infiltrated in the kidneys of patients with IgAN. The intensities of both intrarenal TLR7 and CD19 proteins were closely associated with kidney function (estimated glomerular filtration rate [eGFR] and serum creatinine concentration) and renal histopathology (tubular atrophy, leukocyte infiltration, tubulointerstitial fibrosis, and global glomerulosclerosis) in patients with IgAN. Meanwhile, TLR7 mRNA levels were significantly increased in peripheral blood B cells of patients with IgAN. TLR7+CD19+ B cells expressed inflammatory cytokines (IL-6 and IL-12) in kidneys and produced high levels of IgA1 and galactose deficient-IgA1 (Gd-IgA1) in peripheral blood of patients with IgAN. Mechanistically, TLR7 activated B cells to produce high levels of Gd-IgA1 via the TLR7-GALNT2 axis in IgAN. Protein levels of GALNT2 were increased by overexpression of TLR7, while they were reduced by TLR7 knockdown in B cells. GALNT2 overexpression augmented Gd-IgA1 production in B cells derived from patients with IgAN. Taken together, high TLR7 expression in B cells has dual roles in the development and progression of IgAN, by facilitating renal inflammation and Gd-IgA1 antibody synthesis.

Association between the DOCK7, PCSK9 and GALNT2 Gene Polymorphisms and Serum Lipid levels.

This study was to determine the association between several single nucleotide polymorphisms (SNPs) in the dedicator of cytokinesis 7 (DOCK7), proprotein convertase subtilisin/kexin type 9 (PCSK9) and polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2) and serum lipid levels. Genotyping of 9 SNPs was performed in 881 Jing subjects and 988 Han participants. Allele and genotype frequencies of the detected SNPs were different between the two populations. Several SNPs were associated with triglyceride (TG, rs10889332, rs615563, rs7552841, rs1997947, rs2760537, rs4846913 and rs11122316), high-density lipoprotein (HDL) cholesterol (rs1997947), low-density lipoprotein (LDL) cholesterol (rs1168013 and rs7552841), apolipoprotein (Apo) A1 (rs1997947), ApoB (rs10889332 and rs7552841), and ApoA1/ApoB ratio (rs7552841) in Jing minority; and with TG (rs10889332, rs615563, rs7552841, rs11206517, rs1997947, rs4846913 and rs11122316), HDL cholesterol (rs11206517 and rs4846913), LDL cholesterol (rs1168013), ApoA1 (rs11206517 and rs4846913), ApoB (rs7552841), and ApoA1/ApoB ratio (rs4846913) in Han nationality. Strong linkage disequilibria were noted among the SNPs. The commonest haplotype was G-C-G-C-T-G-C-C-G (>10%). The frequencies of C-C-G-C-T-G-T-C-G, G-C-A-C-T-G-C-C-G, G-C-G-C-T-A-C-C-A, G-C-G-C-T-G-C-C-A, G-C-G-C-T-G-T-C-A haplotypes were different between the two populations. Haplotypes could explain much more serum lipid variation than any single SNP alone especially for TG. Differences in lipid profiles between the two populations might partially attribute to these SNPs and their haplotypes.

Chondroitin sulfate ß-1,4-N-acetylgalactosaminyltransferase-1 (ChGn-1) polymorphism: Association with progression of multiple sclerosis.

Chondroitin sulfate proteoglycans (CSPGs) are a constituent of the matrix of the central nervous system (CNS), likely participating as regulatory molecules in the process of demyelination, remyelination, axonal degeneration and regeneration in the CNS. ChGn-1 is a key enzyme for production of CSPGs and knock-out mice of this gene showed better recovery from spinal cord injury. We hypothesized that the clinical course of multiple sclerosis (MS) is influenced by the level of expression of ChGn-1 gene. We recruited 147 patients with MS and 181 healthy control subjects and analyzed single nucleotide polymorphisms (SNPs) of this gene. We found the coding SNP (cSNP: rs140161612) in approximately 10% of patients with MS as well as normal controls. The cSNP is changed from serine to leucine at position 126 (p.S126L). The expressed ChGn-1 mutant proteins exhibited no enzyme activities in COS-1 cells. In men, patients who had MS with S126L had a slower disease progression. This cSNP might be associated with the sex differences in clinical course of MS.

Probing the O-glycoproteome of gastric cancer cell lines for biomarker discovery.

Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "SimpleCell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer.

Two novel GALNT3 mutations in familial tumoral calcinosis.

Familial tumoral calcinosis (TC) is characterized by elevated serum phosphate concentrations, normal or elevated 1,25(OH)2 vitamin D, as well as periarticular and vascular calcifications. Recessive mutations in the mucin-like glycosyltransferase GalNAc transferase-3 (GALNT3) and the phosphaturic hormone fibroblast growth factor-23 (FGF23) have been shown to result in TC. In the present study, mutational analyses were performed on two patients with TC to determine the molecular basis of their diseases. Analysis of the first patient revealed a novel, homozygous base insertion (1102_1103insT) in GALNT3 exon 5 that results in a frameshift and premature stop codon (E375X). The second patient had a novel homozygous transition (1460 g>a) in GALNT3 exon 7, which caused a nonsense mutation (W487X). Both mutations are predicted to markedly truncate the mature GALNT3 protein product. Although the patients carry GALNT3 mutations, these individuals presented with low-normal serum concentrations of intact biologically active FGF23 and high levels of C-terminal FGF23. In order to discern a possible relationship between GALNT3 and FGF23 in TC, a comprehensive assessment of the reported TC mutations was also performed. In summary, we have detected novel GALNT3 mutations that result in familial TC, and show that disturbed serum FGF23 concentrations are present in our TC cases as well as in previously reported cases. These studies expand our current genetic understanding of familial TC, and support a pathophysiological association between GALNT3 and FGF23.

Serial monitoring of circulating melanoma cells during neoadjuvant biochemotherapy for stage III melanoma: outcome prediction in a multicenter trial.

PURPOSE: Circulating tumor cells (CTCs) in blood may be important in assessing tumor progression and treatment response. We hypothesized that quantitative real-time reverse transcriptase polymerase chain reaction using multimarker mRNA assays could detect CTCs and be used as a surrogate predictor of outcome in patients receiving neoadjuvant biochemotherapy (BC) for melanoma. PATIENTS AND METHODS: Blood specimens were collected at four sampling points from 63 patients enrolled on a prospective multicenter phase II trial of BC before and after surgical treatment of American Joint Committee on Cancer stage III melanoma. Each specimen was assessed by quantitative real-time reverse transcriptase polymerase chain reaction for expression of four melanoma-associated markers: melanoma antigen recognized by T cells 1; beta1 --> 4-N-acetylgalactosaminyltransferase; paired box homeotic gene transcription factor 3; and melanoma antigen gene-A3 family, and the changes of CTCs during treatment and prognostic effect of CTCs after overall treatment on recurrence and survival were investigated. RESULTS: At a median postoperative follow-up time of 30.4 months, 44 (70%) patients were clinically disease free. In relapse-free patients, the number of detected markers significantly decreased during preoperative BC (P = .036), during postoperative BC (P = .002), and during overall treatment (P < .0001). Marker detection after overall treatment was associated with significant decreases in relapse-free and overall survival (P < .0001). By multivariate analysis using a Cox proportional-hazards model, the number of markers detected after overall treatment was a significant independent prognostic factor for overall survival (risk ratio, 12.6; 95% CI, 3.16 to 50.5; P = .0003). CONCLUSION: Serial monitoring of CTCs in blood may be useful for indicating systemic subclinical disease and predicting outcome of patients receiving neoadjuvant BC for metastatic melanoma.

Kinetic studies on Korean serum cis-AB enzymes reveal diminished A and B transferase activities.

BACKGROUND: cis-AB enzymes are rare glycosyltransferases that synthesize both blood group A and B antigens. We have identified a large cohort of Korean cis-AB blood donors and studied the N-acetylgalactosaminyltransferase (glycosyltransferase A, GTA) and galactosyltransferase (glycosyltransferase B, GTB) activity of their cis-AB serum enzymes. MATERIALS AND METHODS: The cis-AB01 allele was identified by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) in 60 donors collected at the Gwangju-Chonnam Red Cross Blood Center. Enzyme assays of this cis-AB enzyme were performed on available serum samples from 16 donors with the cis-AB01/O genotype and three with the cis-AB01/A genotype. RESULTS: In cis-AB donors with an O allele, both the GTA and GTB activity of the cis-AB enzyme were markedly reduced compared to normal A and B controls (29% and 27%, respectively). This is consistent with the behaviour predicted from kinetic studies of a recombinant model of the corresponding AAAB enzyme. CONCLUSION: Although variable, cis-AB enzymes feature reduced GTA and GTB activities. SUMMARY: Cis-AB enzymes feature variable but reduced GTA and GTB activities with relatively weaker GTB activity, consistent with the weak agglutination present on forward typing with anti-B.

Multimarker quantitative real-time PCR detection of circulating melanoma cells in peripheral blood: relation to disease stage in melanoma patients.

BACKGROUND: Detection of melanoma cells in circulation may be important in assessing tumor progression. The objective of this study was to develop a specific, reliable multimarker quantitative real-time reverse transcription-PCR (qRT) assay for detecting melanoma cells in patients' blood. METHODS: We developed qRT assays for the mRNA of four melanoma-associated markers: MART-1, GalNAc-T, PAX-3, and MAGE-A3. In optimization studies, we tested 17 melanoma cell lines and 49 peripheral blood leukocyte (PBL) samples from volunteers. We performed RNA and melanoma cell dilution studies to assess the detection limits and imprecision of the assays. We measured the mRNAs in blood specimens from 94 melanoma patients [American Joint Committee on Cancer (AJCC) stage I, n = 20; II, n = 20; III, n = 32; IV, n = 22]. RESULTS: All markers were frequently detected in melanoma cell lines, whereas none of the markers was detected in PBLs from volunteers. The qRT assay could detect 1 melanoma cell in 10(7) PBLs in the melanoma cell-dilution studies. Markers were detected in 15%, 30%, 75%, and 86% of melanoma patients with AJCC stage I, II, III, and IV disease, respectively. The number of positive markers and AJCC stage were significantly correlated (Spearman correlation coefficient = 0.58; P < 0.0001). CONCLUSIONS: Multimarker qRT can detect circulating melanoma cells in blood. Measurement of the studied molecular markers in blood may be useful in detection of metastasis and monitoring treatment response of melanoma patients.

A novel cis-AB allele derived from the A transferase gene by nucleotide substitution C796A.

The cis-AB is a very rare phenotype in the ABO blood group system. It corresponds to a special ABO allele encoding a glycosyltransferase that is capable of synthesizing both A and B antigens. Until now, gene sequences of only 3 cis-AB alleles were characterized. One was the A(1v) allele with a nucleotide substitution G803C at codon 268; the second was the B allele with a nucleotide substitution A796C at codon 266; and the third arose from a point mutation C700T at codon 234 in exon 7 of the B transferase gene. In this study, we found a novel cis-AB allele when performing paternity tests in Chang Gung Memorial Hospital in Taiwan. Although his father was O blood type, a serologically AB blood type child was confirmed as being his father's offspring on the basis of 16 microsatellite markers (99.97% plausibility for the child and father). Exons 6 and 7 of the child's ABO alleles were characterized by direct sequencing and gene cloning. The results showed that the child has one O(1) allele and the second allele is almost identical to A(1*02) allele except for a single point mutation at nucleotide position 796, where an A replaces a C and leads to a change of leucine to methionine at amino acid 266. This implies that the child's O(1) allele was inherited from his father and the other allele was inherited from his mother. In conclusion, the novel cis-AB allele reported here is derived from the A transferase gene through a nucleotide substitution C796A, which differs from the 3 previously reported cis-AB alleles.

Detection procedures for neuroblastoma cells metastatic to blood and bone marrow: blinded comparison of chromogranin A heminested reverse transcription polymerase chain reaction to tyrosine hydroxylase nested reverse transcription polymerase chain reaction and to anti-GD2 immunocytology.

Specific and sensitive tumor cell detection is becoming increasingly important for diagnosing and staging as well as for the therapeutic management of neuroblastoma patients. We propose a chromogranin A heminested reverse transcription polymerase chain reaction (CgA hn RT-PCR) procedure for the detection of neuroblastoma minimal residual disease in peripheral blood and bone marrow samples. The results were checked in comparison with the presently available procedures (i.e., with the tyrosine hydroxylase nested RT-PCR [TH n RT-PCR] and with the immunocytochemical approach using anti-GD2 antibodies). Controls from healthy patients or from people with unrelated disease (12 samples of bone marrow and 23 samples of peripheral blood) and serial dilution experiments using neuroblastoma cell lines (SKNLP, SKNFI, STA6, STA8) showed CgA hn RT-PCR full specificity and sensitivity ranging from 10(3) to 10(6) (depending on the cell line). The results compared favorably with those obtained using TH n RT-PCR. Preliminary data obtained analyzing bone marrow and peripheral blood specimens from stage IV neuroblastomas showed substantially overlapping results between CgA and TH n RT-PCR procedures. Our data support the potential usefulness of CgA heminested RT-PCR as a specific and sensitive procedure for minimal disease detection in neuroblastoma. A prospective evaluation of this tool in clinical studies might be warranted.

Detection of occult metastatic breast cancer cells in blood by a multimolecular marker assay: correlation with clinical stage of disease.

Currently, molecular markers offer the unique opportunity to identify occult metastasis in early stage cancer patients not otherwise detected with conventional staging techniques. To date, well-characterized molecular tumor markers to detect occult breast cancer cells in blood are limited. Because breast tumors are heterogeneous in tumor marker expression, we developed a "multimarker" reverse transcription-PCR assay combined with the highly sensitive electrochemiluminescence automated detection system. Breast cancer cell lines (n = 7), primary breast tumors (n = 25), and blood from normal donors (n = 40) and breast cancer patients [n = 65; American Joint Committee on Cancer (AJCC) stages I-IV] were assessed for four mRNA tumor markers: beta-human chorionic gonadotropin (beta-hCG), oncogene receptor (c-Met), beta 1-->4-N-acetylgalactosaminyl-transferase, and a tumor-associated antigen (MAGE-A3). None of the tumor markers were expressed in any normal donor bloods. Breast cancer cell lines and primary breast tumors expressed beta-hCG, c-Met, beta 1-->4-N-acetylgalactosaminyl-transferase, and MAGE-A3 mRNA. Of the 65 breast cancer patient blood samples assessed, 2, 3, 15, 49, and 31% expressed 4, 3, 2, 1, and 0 of the mRNA tumor markers, respectively. At least two markers were expressed in 20% of the blood specimens. The addition of a combination of markers enhanced detection of systemic metastasis by 32%. In patient blood samples, the MAGE-A3 marker correlated significantly with tumor size (P = 0.0004) and AJCC stage (P = 0.007). The combination of beta-hCG and MAGE-A3 mRNA markers correlated significantly with tumor size (P = 0.04), and the marker combination c-Met and MAGE-A3 showed a significant correlation with tumor size (P = 0.005) as well as AJCC stage (P = 0.018). A multimarker reverse transcription-PCR assay that correlates with known clinicopathological prognostic parameters may have potential clinical utility by monitoring tumor progression with a blood test.

Purification and characterization of fetal bovine serum beta-N-acetyl-D-galactosaminyltransferase and beta-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis.

beta-N-Acetylgalactosaminyltransferase II and beta-glucuronyltransferase II, involved in chondroitin sulfate biosynthesis, transfer an N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) residue, respectively, through beta-linkages to an acceptor chondroitin oligosaccharide derived from the repeating disaccharide region of chondroitin sulfate. They were copurified from fetal bovine serum approximately 2500-fold and 850-fold, respectively, by sequential chromatographies on Red A-agarose, phenyl-Sepharose, S-Sepharose and wheat germ agglutinin-agarose. Identical and inseparable chromatographic profiles of both glycosyltransferase activities obtained through the above chromatographic steps and gel filtration suggest that the purified enzyme activities are tightly coupled, which could imply a single enzyme with dual transferase activities; beta-N-acetylgalactosaminyltransferase and beta-glucuronyltransferase, reminiscent of the heparan sulfate polymerase reaction. However, when a polymerization reaction was performed in vitro with the purified serum enzyme preparation under the polymerization conditions recently developed for the chondroitin-synthesizing system, derived from human melanoma cells, each monosaccharide transfer took place, but no polymerization occurred. These results may suggest that the purified serum enzyme preparation contains both beta-N-acetylgalactosaminyltransferase II and beta-glucuronyltransferase II activities on a single polypeptide or on the respective polypeptides forming an enzyme complex, but is different from that obtained from melanoma cells in that it transfers a single GalNAc or GlcA residue but does not polymerize chondroitin.

Regulation of chondroitin sulfate biosynthesis by specific sulfation: acceptor specificity of serum beta-GalNAc transferase revealed by structurally defined oligosaccharides.

The relationship between sulfation and polymerization in chondroitin sulfate (CS) biosynthesis has been poorly understood. In this study, we investigated the specificity of bovine serum UDP-GalNAc: CS beta-GalNAc transferase responsible for chain elongation using structurally defined acceptor substrates. They consisted of tetra- and hexasaccharide-serines that were chemically synthesized and various regular oligosaccharides with a GlcA residue at the nonreducing terminus, prepared from chondroitin and CS using testicular hyaluronidase. The enzyme preparation was obtained from fetal bovine serum by means of heparin-Sepharose affinity chromatography. The preparation did not contain the alpha-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995), that utilizes common acceptor substrates. The beta-GalNAc transferase used as acceptors, two hexasaccharide-serines GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal (4-sulfate) beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, but neither the monosulfated hexasaccharide-serine GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser nor tetrasaccharide-serines with or without a sulfate group at C-4 of the third sugar residue Gal-3 from the reducing end. The results indicated that the sulfate group at the Gal-3 C-4 markedly affected the transfer of GalNAc to the terminal GlcA. In addition, a sulfate group at C-4 of the reducing terminal GalNAc of regular tetrasaccharides remarkably enhanced the GalNAc transfer, suggesting that the enzyme recognizes up to the fourth saccharide residue from the nonreducing end. The level of incorporation into a tetra- or hexasaccharide containing a terminal 2-O-sulfated GlcA residue was significant, whereas there was no apparent incorporation into tetra- or hexasaccharides containing a terminal 3-O-sulfated GlcA or penultimate 4,6-O-disulfated GalNAc residue. These results indicated that sulfation reactions play important roles in chain elongation and termination.

Appearance of beta 1,4 N-acetylgalactosaminyltransferase (glycosphingolipids GA2/GM2/GD2 synthase) in embryonic chicken vitreous humor during development.

PURPOSE: beta 1,4 N-Acetylgalactosaminyltransferase (GalNAcT) is a type II integral membrane protein of the Golgi apparatus that catalyzes the synthesis of the glycosphingolipids GM2, GD2, and GA2. The activity of GalNAcT in chick retinal cells increases 6-fold between embryonic days 7 and 14. Because GalNAcT, like many Golgi glycosyltransferases, is proteolytically cleaved from Golgi membranes to release a soluble form into the culture medium of cells transfected with the cloned human enzyme, we tested whether GalNAcT might be released from embryonic retinal cells into the vitreous humor. METHODS: Samples of vitreous humor and plasma and extracts of retinal cells were assayed for GalNAcT activity. RESULTS: The activity of a soluble form of GalNAcT in embryonic chick vitreous humor was nearly undetectable until embryonic day 10, then increased more than six fold until day 16, and remained at that level until birth. The activity was identified as authentic GalNAcT based on a requirement for Mn++, GSL substrate specificity, and product characterization. GalNAcT activity in embryonic plasma was roughly 10% that of the corresponding vitreous humor, suggesting that the plasma was not the source of the activity in the vitreous. CONCLUSIONS: GalNAcT in embryonic chicken vitreous humor is likely due either to a specific release from neural retinal cells or due to non-specific lysis of these cells during apoptosis associated with the development of the retina. Regardless of the source, GalNAcT in the vitreous humor has the potential to function as a lectin by binding to gangliosides GD3 and GM3 on the surface of retinal cells and, thereby, to influence neuronal development.

Can blood group O red cells of donor origin acquire weak group A reactivity through serum A transferase of the recipient after bone marrow transplantation?

BACKGROUND: Blood group A substance was detected on red cells of a patient who received a bone marrow transplant from a blood group O donor 3.5 years ago. MATERIALS AND METHODS: Peripheral blood was investigated by conventional serological techniques, fluorescence in situ hybridisation, and polymerase chain reaction. RESULTS: All peripheral blood cells are of donor origin. Anti-A and not anti-A, B of blood group O individuals can be absorbed to the group O red cells of the patient. CONCLUSION: We suppose that the patient's residual serum A transferase attaches the appropriate sugar to substance H on the red cell membrane to form substance A.

N-acetylgalactosamine (GalNAc) transfer to the common carbohydrate-protein linkage region of sulfated glycosaminoglycans. Identification of UDP-GalNAc:chondro-oligosaccharide alpha-N-acetylgalactosaminyltransferase in fetal bovine serum.

During the course of a study of elucidate the role of modification of the common polysaccharide-protein linkage structure, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, in biosynthetic sorting mechanisms of the different sulfated glycosaminoglycan chains, a novel N-acetylgalactosamine (GalNAc) transferase was discovered in fetal bovine serum. The enzyme catalyzed the transfer of [3H]GalNAc from UDP-[3H]GalNAc to linkage tetrasaccharide and hexasaccharide serines synthesized chemically and to various regular oligosaccharides containing terminal D-glucuronic acid (GlcA), which were prepared from chondroitin and chondroitin sulfate using testicular hyaluronidase digestion. The labeled products obtained with the linkage tetra- and hexasaccharide serines and with the tetrasaccharide (GlcA beta 1-3GalNAc)2 were resistant to digestion with chondroitinase AC-II and beta-N-acetylhexosaminidase but sensitive to alpha-N-acetylgalactosaminidase digestion, indicating that the enzyme is an alpha-N-acetylgalactosaminyltransferase. This finding is in contrast to that of Rohrmann et al. (Rohrmann, K., Niemann, R., and Buddecke, E. (1985) Eur. J. Biochem., 148, 463-469), who reported that a corresponding product was susceptible to digestion with beta-N-acetylhexosaminidase. The presence of a sulfate group at C4 of the penultimate GalNAc or Gal units markedly inhibited the transfer of GalNAc to the terminal GlcA, while a sulfate group at C6 of the GalNAc had little effect on the transfer. Moreover, a slight but significant transfer of [3H]GalNAc was observed to an oligosaccharide containing terminal 2-O-sulfated GlcA as acceptor, whereas no incorporation was detected into oligosaccharides containing terminal unsaturated or 3-O-sulfated GlcA units. These results suggest that this novel serum enzyme is a UDP-GalNAc:chondro-oligosaccharide alpha 1-3- or 1-4-N-acetylgalactosaminyltransferase. The possibility of involvement of this enzyme in glycosaminoglycan biosynthesis is discussed.

Detection and characterization of UDP-GalNAc: chondroitin N-acetylgalactosaminyltransferase in bovine serum using a simple assay method.

We report the occurrence, in bovine serum of a soluble form, of N-acetylgalactosaminyl-transferase activity, which is involved in chondroitin sulfate biosynthesis, and we describe a simple assay method for the enzyme. Chondroitin with the general structure [GlcA-GalNAc], was found to serve as acceptor substrates for the enzyme. To develop a routine assay, bovine serum was incubated with polymeric chondroitin and UDP-[3H]GalNAc. After the removal of labeled endogenous acceptors derived from serum by trichloroacetic acid treatment, the labeled chondroitin sulfate chains were separated from residual unincorporated label by centrifugation using syringe columns packed with Sephadex G-25, and the effluent fractions were monitored by scintillation counting. This simple method allowed us to perform multiple assays and to establish assay conditions for the serum N-acetylgalactosaminyltransferase involved in chondroitin polymerization.