Studies on the structure and I-blood-group activity of poly(glycosyl)ceramides.
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Employing a modified technique of acetolysis, which allows almost a complete recovery of constituent sugars from poly(glycosyl)ceramides, the glycolipids were found to contain an excess of N-acetylglucosamine over galactose. On the basis of Smith degradation, methylation study, chromium trioxide degradation and the structures of oligosaccharides released from the glycolipids by partial acid hydrolysis, the presence of two types of sugar sequences has been established in poly(glycosyl)ceramides: a) Galbeta1 leads to 4GlcNAcbeta1 leads to 6Gal3 comes from R1 b) Galbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc1 leads to R2. The repeating unit of poly(glycosyl)ceramides seems to be the GlcNAcbeta1 leads to 3Gal sequence. The specificity of one anti-I serum (Woj) is directed against the non-reducing ending of the first kind of chain. Three other anti-I sera reacted with inner portions of the oligosaccharide chains of the glycolipids. (+info)
Molecular basis of the adult i phenotype and the gene responsible for the expression of the human blood group I antigen.
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The human blood group i and I antigens are characterized as linear and branched repeats of N-acetyllactosamine, respectively. Conversion of the i to the I structure requires the activity of I-branching beta-1,6-N-acetylglucosaminyltransferase (IGnT). Thus the blood group I gene is assigned to encode a beta-1,6-N-acetylglucosaminyltransferase; however, its identity has not been confirmed. The null phenotype of I, the adult i phenotype, provides a means to identify the I gene. Interestingly, the adult i phenotype has been noted to be associated with congenital cataracts in Asians. Molecular genetic studies of 3 adult i pedigrees are reported here. The results obtained on mutation detection within the 2 I-branching enzyme encoding genes, segregation analyses, and enzyme function assays identify molecular changes associated with the adult i phenotype. The adult i phenotype in 2 of the pedigrees studied resulted from 1043G-->A and 1148G-->A mutations, which predict Gly348Glu and Arg383His alterations, respectively, in the IGnT gene. These amino acid changes abolished the original GlcNAc-transferase activity. Deletion of the IGnT gene was observed in the person with adult i phenotype in the third pedigree. These findings suggest that the IGnT gene, first reported in 1993, is the candidate for the blood group I gene. Confirmation of the blood group I gene will further assist in the investigations of the molecular genetics that control I antigen expression in secretions and the molecular basis for the association of the adult i phenotype with congenital cataracts in Asians. (+info)
The molecular genetics of the human I locus and molecular background explain the partial association of the adult i phenotype with congenital cataracts.
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The human i and I antigens are characterized as linear and branched repeats of N-acetyllactosamine, respectively. Conversion of the i to the I structure requires I-branching beta-1,6-N-acetylglucosaminyltransferase activity. It has been noted that the null phenotype of I, the adult i phenotype, is associated with congenital cataracts in Asians. Previously, the identification of molecular changes in the IGnT gene, associated with the adult i phenotype, has been reported. In the present study, we demonstrate that the human I locus expresses 3 IGnT forms, designated IGnTA, IGnTB, and IGnTC, which have different exon 1, but identical exons 2 and 3, coding regions. The molecular genetics proposed for the I locus offer a new perspective on the formation and expression of the I antigen in different cells and provide insight into the questions derived from investigation of the adult i phenotype. Molecular genetic analyses of the I loci of the 2 adult i groups, with and without congenital cataracts, were performed, and enzyme function assays and expression patterns for the 3 IGnT transcripts in reticulocytes and lens-epithelium cells were analyzed. The results suggest a molecular genetic mechanism that may explain the partial association of the adult i phenotype with congenital cataracts and indicate that a defect in the I locus may lead directly to the development of congenital cataracts. The results also suggest that the human blood group I gene should be reassigned to the IGnTC form, not the IGnTB form, as described previously. (+info)
A novel I-branching beta-1,6-N-acetylglucosaminyltransferase involved in human blood group I antigen expression.
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The human blood group i and I antigens are determined by linear and branched poly-N-acetyllactosamine structures, respectively. In erythrocytes, the fetal i antigen is converted to the adult I antigen by I-branching beta-1,6-N-acetylglucosaminyltransferase (IGnT) during development. Dysfunction of the I-branching enzyme may result in the adult i phenotype in erythrocytes. However, the I gene responsible for blood group I antigen has not been fully confirmed. We report here a novel human I-branching enzyme, designated IGnT3. The genes for IGnT1 (reported in 1993), IGnT2 (also presented in this study), and IGnT3 consist of 3 exons and share the second and third exons. Bone marrow cells preferentially expressed IGnT3 transcript. During erythroid differentiation using CD34(+) cells, IGnT3 was markedly up-regulated with concomitant decrease in IGnT1/2. Moreover, reticulocytes expressed the IGnT3 transcript, but IGnT1/2 was below detectable levels. By molecular genetic analyses of an adult i pedigree, individuals with the adult i phenotype were revealed to have heterozygous alleles with mutations in exon 2 (1006G>A; Gly336Arg) and exon 3 (1049G>A; Gly350Glu), respectively, of the IGnT3 gene. Chinese hamster ovary (CHO) cells transfected with each mutated IGnT3 cDNA failed to express I antigen. These findings indicate that the expression of the blood group I antigen in erythrocytes is determined by a novel IGnT3, not by IGnT1 or IGnT2. (+info)
Immunoelectron microscopic studies reveal differences in distribution of sialo-oligosaccharide receptors for Mycoplasma pneumoniae on the epithelium of human and hamster bronchi.
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Long-chain sialo-oligosaccharides with poly-N-acetyllactosamine backbones (Ii antigen type) are major host cell receptors for the human pathogen Mycoplasma pneumoniae. Previous immunofluorescence studies of the human bronchial epithelium, using sequence-specific monoclonal antibodies to the branched I-type and linear i-type backbones, have indicated that sialylated and nonsialylated long-chain sequences of both types are richly expressed on the ciliated cells, where they are polarized at the apical aspects. These sequences are lacking in the goblet cells. In the present study, the display of these oligosaccharides has been investigated by electron microscopy (immunogold labelling) in the human bronchial epithelium and in that of the hamster, an animal model commonly used for M. pneumoniae infection. In the human bronchial epithelium, the long-chain branched sequences have been detected along the entire length of the cilia and on microvilli, whereas the linear sequences are confined to the microvilli and the basal aspects of the cilia. On the ciliated epithelial cells of the hamster, by contrast, the branched and linear sequences (sialo- and asialo-) have been detected exclusively on microvilli. A further striking difference is that in the hamster these structures are expressed in abundance on the goblet cells and in the intracellular globules. We suggest that the latter finding may partly explain the relatively large doses of M. pneumoniae required to establish experimental infection in the hamster, as the receptor-bearing secreted mucus may have a protective role in binding to the microorganisms, leading to their clearance by bronchociliary action. (+info)
A nonsense mutation in the glucosaminyl (N-acetyl) transferase 2 gene (GCNT2): association with autosomal recessive congenital cataracts.
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PURPOSE: To identify the genetic defect associated with autosomal recessive congenital cataract in four Arab families from Israel. METHODS: Genotyping was performed using microsatellite markers spaced at approximately 10 cM intervals. Two-point lod scores were calculated using MLINK of the LINKAGE program package. Mutation analysis of the glucosaminyl (N-acetyl) transferase 2 gene (GCNT2) gene was performed by direct sequencing of PCR-amplified exons. RESULTS: The cataract locus was mapped to a 13.0-cM interval between D6S470 and D6S289 on Chr. 6p24. A maximum two-point lod score of 8.75 at theta = 0.019 was obtained with marker D6S470. Sequencing exons of the GCNT2 gene, mutations of which have been associated with cataracts and the i blood group phenotype, revealed in these families a homozygous G-->A substitution in base 58 of exon-2, resulting in the formation of premature stop codons W328X, W326X, and W328X, of the GCNT2A, -B, and -C isoforms, respectively. Subsequent blood typing of affected family members confirmed the possession of the rare adult i blood group phenotype. CONCLUSIONS: A nonsense mutation in the GCNT2 gene isoforms is associated with autosomal recessive congenital cataract in four distantly related Arab families from Israel. These findings provide further insight into the dual role of the I-branching GCNT2 gene in the lens and in reticulocytes. (+info)
Biosynthesis of blood group I and i antigens in rat tissues. Identification of a novel beta 1-6-N-acetylglucosaminyltransferase.
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The beta-galactoside beta 1-6- and beta 1-3-N-acetylglucosaminyltransferases (beta 1-6GnT and beta 1-3GnT) that synthesize blood group I and i antigens were identified in rat tissues, using pyridylaminated lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-PA) as an acceptor. The products of the transferase reactions were separated on high performance liquid chromatography. The products of the transferase reactions were identified by 1H NMR as (formula; see text) and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-PA. The product for beta 1-6GnT was also identified by methylation analysis. Kinetic experiments were carried out using rat serum for beta 1-3GnT and partially purified enzyme from rat intestine for beta 1-6GnT. beta 1-3GnT has a pH optimum of 7.5 and requires Mn2+ for optimal activity. beta 1-6GnT has a pH optimum of 7.0 and does not require Mn2+. Studies on the substrate specificity of each enzyme indicated that the preferred substrate for beta 1-3GnT had the general structure Gal beta 1-4GlcNAc-OR and, for beta 1-6GnT, Gal beta 1-4GlcNAc beta 1-3Gal-OR where R = sugar. This is the first demonstration that the beta 1-6GnT acts on an internal galactose of lacto-N-neotetraose and paragloboside, and the enzyme appears to be a novel enzyme in terms of substrate specificity. (+info)
Variable region gene analysis of pathologic human autoantibodies to the related i and I red blood cell antigens.
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To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B-cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline-encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti-I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens. (+info)