Purification of N-acetyllactosamine-binding activity from the porcine sperm membrane: possible involvement of an ADAM complex in the carbohydrate-binding activity of sperm.

Although the importance of carbohydrate recognition by sperm during egg zona pellucida binding has been widely reported, the sperm molecular species that recognize the carbohydrates are poorly characterized. Our previous cytochemical study indicated that two kinds of carbohydrate-binding proteins are expressed on porcine sperm heads-one recognizes N-acetyllactosamine (Galß1-4GlcNAc-), and the other recognizes the Lewis X structure (Galß1-4(Fucα1-3)GlcNAc-). For this report, we used proteomic techniques to characterize the sperm proteins that bind N-acetyllactosamine. Porcine sperm plasma membrane was solubilized with a detergent solution and subjected to sequential chromatography with dextran sulfate agarose, affinity, and hydroxyapatite, and the binding activities in the eluates were monitored by a solid-phase binding assay. The tryptic peptides of two proteins most likely associated with the binding activities were subjected to tandem mass spectrometry sequencing. A subsequent database search identified one of the two proteins as predicted disintegrin and metalloprotease domain-containing protein 20-like (XP_003128672). The other protein was identified as disintegrin and metalloprotease domain-containing protein 5 (AB613817) by database searches for homologous amino acid sequences, cDNA cloning, nucleotide sequencing and nucleotide database searches. Furthermore, two-dimensional blue native/SDS-PAGE demonstrated that they formed a variety of non-covalent complexes. Therefore, these ADAM complexes probably are responsible for the N-acetyllactosamine-binding activity. An affinity-purified fraction containing these ADAM complexes showed zona pellucida-binding activity, though the activity was relatively weak, and the presence of another zona pellucida-binding protein that probably works in concert with these ADAM complexes was suggested. Immunofluorescence testing suggested that ADAM20-like was localized on the anterior part of the sperm plasma membrane.

The analysis of N-glycolylneuraminic acid(NeuGc) of hepatoma tissue and K562 cell ferritins using HPLC and mass spectrometry.

Ferritin is an iron-storage protein and its serum level is known to increase in the patient of with inflammation and malignant tumor. To further elucidate the difference between ferritins from normal human liver tissue and that of cancer cells, their sialic acids were analyzed. The Western blot analysis and the cytochemical staining using anti-NeuGc antiserum indicated that ferritins from the human hepatocarcinoma tissue and malignant K562 cells contain NeuGc, but that from the normal liver does not. The result was also confirmed by HPLC analysis and MALDI-TOF/MS analysis of sialic acids which were derivatized by the DMB method. It was also shown that the sialic acid content in hepatocarcinoma ferritin was much higher than that in the normal liver ferritin. These results suggest that normal and cancerous liver ferritins are qualitatively and quantitatively different in sialylation. In addition, K562 cells were shown to express NeuGc even if the cells were cultured in serum-free media which lack NeuGc. This is of interest from the current concept that expression of NeuGc in human cells is due to uptake and utilization of exogenous NeuGc.

Therapeutic effects of novel anti-tumor reagent, apoptosis inducing nucleosides from CD57+HLA-DRbright natural suppressor cell line on human gastric carcinoma-bearing SCID mice.

To further provide scientific evidence before clinical application, the anti-tumor effects of apoptosis inducing nucleosides (AINs) released from CD57+HLA-DRbright natural suppressor (CD57.DR-NS) cell line on human gastric carcinoma (GCIY)-bearing severe combined immunodeficiency (SCID) mice were examined by monitoring tumor cell growth and change of body weight of mice. The results obtained evidenced that AINs strongly induced apoptosis in the tumor tissues in SCID mice with decrease of tumor size and without loss of body weight. We found that peak 5 and peak 6 (P5 and P6) components among six components (AINs) isolated from CD57.DR-NS cell cultures by high performance liquid chromatography (HPLC) are the most effective. The anti-tumor effective dosage of P5, P6 and their mixture, P5+P6, were obtained in dose-dependent manner. Thus, the most effective method of administration of AINs for tumor regression without exhaustion was established in the present study. Corresponding to the previous study that AINs could generate apoptosis in malignant cells while lacking the toxicity in normal cells, the results obtained in the present preclinical experiments suggested anti-tumor efficacy of AINs with possible refrainment from side-effects in clinical trials.

Polylactosamine synthesis and branch formation of N-glycans in beta1,4-galactosyltransferase-1-deficient mice.

Analysis of glycans from erythrocyte membrane glycoproteins from beta1,4-galactosyltransferase-1 (beta4GalT-1)-deficient mice revealed moderately decreased galactosylation but comparable polylactosamine content compared to control beta4GalT-1(+/-) mice. The increased expression of more branched N-glycans was observed in beta4GalT-1(-/-) mice, and its extent was more remarkable in elder beta4GalT-1(-/-) mice (28 weeks old) than in younger beta4GalT-1(-/-) mice (6-9 weeks old). In relation to this issue, the less galactosylation of biantennary glycans was observed in the elder group, suggesting that beta4GalTs actually compete with N-acetylglucosaminyltransferases IV and V in erythroid cells. In contrast, approximately 80% of core 2 O-glycans were not beta1,4-galactosylated regardless of age of the knockout mice. These results suggest that beta4GalT-1 expressed in erythroid cells may regulate a constant branch formation of N-glycans and plays a predominant role in beta1,4-galactosylation of core 2 O-glycan.

Glycophorin A requirement for expression of O-linked antigens on the erythrocyte membrane.

BACKGROUND: Glycophorin A (GPA) has a large number of sialic acid-containing oligosaccharide chains. GPA is highly conserved among vertebrates, mice with a GPA deletion have not been reported and GPA's physiologic role remains uncertain. RESULTS: GPA-/- homozygotes were obtained by intercrossing GPA+/- heterozygotes based on Mendelian genetics. The amount of O-linked oligosaccharide chains in the erythrocyte membrane of GPA-/- mice decreased to 60% compared to that of the wild-type mice. Flow cytometry and Western blot analysis revealed that the TER antigen that is associated with GPA on the erythrocyte membrane was totally abrogated from the cell surface in GPA-/- mice. Several glycoproteins that were detected with peanut agglutinin (PNA), a lectin that recognizes O-linked oligosaccharide chains, were absent from the GPA-/- erythrocyte membrane. Erythrocytes lacking GPA were more sensitive to hypo-osmotic stress than wild-type erythrocyte. CONCLUSIONS: GPA-/- mice show apparently normal phenotypes at least during the early generations. The disappearance of many glycoproteins recognized by PNA lectin on the GPA-/- erythrocyte membrane proteins suggests that GPA has an essential role in the expression of O-linked antigens on the erythrocyte membrane protein. These interactions of GPA and other glycoproteins may contribute to maintaining the physical strength of the erythrocyte membrane.

Aberrant O-glycosylation inhibits stable expression of dysadherin, a carcinoma-associated antigen, and facilitates cell-cell adhesion.

Recently, we identified dysadherin, a novel carcinoma-associated glycoprotein, and showed that overexpression of dysadherin in human hepatocarcinoma PLC/PRF/5 cells could suppress E-cadherin-mediated cell-cell adhesion and promote tumor metastasis. The present study shows evidence that dysadherin is actually O-glycosylated. This was based on a direct carbohydrate composition analysis of a chimera protein of an extracellular domain of dysadherin fused to an Fc fragment of immunoglobulin. To assess the importance of O-glycosylation in dysadherin function, dysadherin-transfected hepatocarcinoma cells were cultured in a medium containing benzyl-alpha-GalNAc, a modulator of O-glycosylation. This treatment facilitated homotypic cell adhesion among dysadherin transfectants accompanied with morphological changes, indicating that the anti-adhesive effect of dysadherin was weakened. Modification of O-glycan synthesis also resulted in down-regulation of dysadherin expression and up-regulation of E-cadherin expression in dysadherin transfectants but did not affect E-cadherin expression in mock transfectants. Structural analysis of O-glycans released from the dysadherin chimera proteins indicated that a series of O-glycans with core 1 and 2 structures are attached to dysadherin, and their sialylation is remarkably inhibited by benzyl-alpha-GalNAc treatment. However, sialidase treatment of the cells did not affect calcium-dependent cell aggregation, which excluded the possibility that sialic acid itself is directly involved in cell-cell adhesion. We suggest that aberrant O-glycosylation in carcinoma cells inhibits stable expression of dysadherin and leads to the up-regulation of E-cadherin expression by an unknown mechanism, resulting in increased cell-cell adhesion. The carbohydrate-directed approach to the regulation of dysadherin expression might be a new strategy for cancer therapy.

Impaired selectin-ligand biosynthesis and reduced inflammatory responses in beta-1,4-galactosyltransferase-I-deficient mice.

Selectins recognize ligands containing carbohydrate chains such as sialyl Lewis x (sLex) that are mainly presented at the terminus of N-acetyl lactosamine repeats on core 2 O-glycans. Several glycosyltransferases act successively to extend the N-acetyl lactosamine repeats and to synthesize sLex, and beta-1,4-galactosyltransferase (beta4GalT) plays a key role in these processes. Recently isolated 6 beta4GalT genes are candidates, but their individual roles, including those in selectin-ligand biosynthesis, remain to be elucidated. More than 80% of the core 2 O-glycans on the leukocyte membrane glycoproteins of beta4GalT-I-deficient mice lacked galactose residues in beta-1,4 linkage, and soluble P-selectin binding to neutrophils and monocytes of these mice was significantly reduced, indicating an impairment of selectin-ligand biosynthesis. beta4GalT-I-deficient mice exhibited blood leukocytosis but normal lymphocyte homing to peripheral lymph nodes. Acute and chronic inflammatory responses, including the contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) responses, were suppressed, and neutrophil infiltration into inflammatory sites was largely reduced in these mice. Our results demonstrate that beta4GalT-I is a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of beta4GalT-I-deficient mice are impaired because of the defect in selectin-ligand biosynthesis.