Sialidase Digestion Research Articles

Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells

Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.

Novel O-linked glycans containing 6'-sulfo-Gal/GalNAc of MUC1 secreted from human breast cancer YMB-S cells: Possible carbohydrate epitopes of KL-6(MUC1) monoclonal antibody

Human serum Krebs von den Lugen-6 (KL-6) antigen is a MUC1 glycoprotein (KL-6/MUC1) recognized by anti-KL-6 monoclonal antibody (KL-6/mAb) and has been utilized as a diagnostic marker for interstitial pneumonia. KL-6/mAb is thought to recognize the specific glycopeptides sequence of MUC1, but the precise glycan structure of the epitope is unclear. In this study, we determined the carbohydrate structures of KL-6/MUC1 to search the carbohydrate epitopes for KL-6/mAb. KL-6/MUC1 was purified from the culture medium of human breast cancer YMB-S cells by KL-6/mAb-affinity chromatography; the O-linked glycan structures were determined in combination with paper electrophoresis, several lectin column chromatographies, sialidase digestion and methanolysis. KL-6/MUC1 contained core 1 and extended core 1 glycans modified with one or two sialic acid/sulfate residues. Based on these structures, several synthetic glycans binding to anti-KL-6/mAb were compared with one another by surface plasmon resonance. Sequentially, related radiolabeled oligosaccharides were enzymatically synthesized and analyzed for binding to a KL-6/mAb-conjugated affinity column. 3'-sialylated, 6'-sulfated LNnT [Neu5Acα2-3(SO(3)(-)-6)Galβ1-4GlcNAcβ1-3Galβ1-4Glc], 3'-sialylated, 6-sulfated core 1 [Neu5Acα2-3Galβ1-3(SO(3)(-)-6)GalNAc] and disulfated core 1 SO(3)(-)-3Galβ1-3(SO(3)(-)-6)GalNAc exhibited substantial affinity for KL-6/mAb, and 3'-sulfated core 1 derivatives [SO(3)(-)-3Galβ1-3(±Neu5Acα2-6)GalNAc] and 3'-sialylated core 1 weakly interacted with KL-6/mAb. These results indicated that the possible carbohydrate epitopes of KL-6/mAb involve not only 3'-sialylated core 1 but also novel core 1 and extended core 1 with sulfate and sialic acid residues. Epitope expressing changes with suppression or over-expression of the Gal6ST (Gal 6-O-sulfotransferase) gene, suggesting that Gal6ST is involved in the biosynthesis of the unique epitopes of KL-6/mAb.

Histochemical Detection of Glycoconjugates in the Inner Perivitelline Layer of Japanese Quail (Coturnix japonica)

The avian inner perivitelline layer (IPVL), a homologous structure to the mammalian zona pellucida, is deposited between the granulosa cells and the oocyte cell membrane during folliculogenesis. In this glycohistochemical study, a panel of fluorescein isothiocyanate (FITC)-labelled lectins was used to characterise and localise the oligosaccharide sequences of the IPVL glycoproteins at different stages of follicular development in the quail ovary. Deacetylation and sialidase digestion were also performed prior to lectin cytochemistry. Contrary to mammals, where the topographical distribution of these carbohydrates is not uniformly distributed throughout the zona pellucida, indicating the regionalisation of oligosaccharide chains, our results demonstrated a homogenous lectin staining of the comparatively thin IPVL. We also found variations in the presence and distribution of the carbohydrate residues in the IPVL during different stages of follicular growth. The IPVL of pre-vitelline follicles distinctly stains with WGA, sWGA and SBA, demonstrating the presence of D-GlcNAc, Neu5Ac and α-D-GalNac in the glycoproteins of the forming IPVL. No staining was found with ConA (specific for α-D-Man, α-D-Glc), LCA (α-D-Man, α-D-Glc), PNA (β-D-Gal-(1-3)-D-GalNAc, VAA (Gal), DBA (α-D-GalNAc(1-3)-GalNAc and UEA-I (α-L-Fuc). With continuing follicular growth of the oocyte and the follicle, this staining pattern changed. LCA and PNA-staining in the IPVL became distinctly positive. As the IPVL of immature oocytes distinctly stains with WGA/sWGA-FITC, but spermatozoa do not bind to immature zona, it appears questionable that carbohydrate residues detected by WGA/sWGA play a major role in sperm-IPVL binding, as suggested in previous investigations.

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4–7 N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

BackgroundDentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties.ResultsTo identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr200, Thr216 and Thr316. Porcine Dsp GAG attachments were found at Ser238 and Ser250 and were comprised of chondroitin 6-sulfate and chondroitin 4-sulfate in a ratio of 7 to 3, respectively.ConclusionsThe distribution of porcine Dsp posttranslational modifications indicate that porcine Dsp has an N-terminal domain with at least six N-glycosylations and a C-terminal domain with two GAG attachments and at least two O-glycosylations.

Lectin-binding pattern of Senegalese sole Solea senegalensis (Kaup) testis.

The localization and characterization of oligosaccharide sequences in the testis of Senegalese sole Solea senegalensis was investigated using 12 lectins in combination with KOH saponification and sialidase digestion (K-s). The interstitial compartment contained all the sugar residues investigated, those bearing oligosaccharides terminating with sialic acid (Neu5Ac) alpha2,3Galbeta1,4GlcNAc, Neu5AcGalNAcalpha1,3(LFucbeta1,2) Galbeta1,3/4GlcNAcbeta1 and GalNAcalpha1,3(LFuc1,2) Galbeta1,3/4GlcNAcbeta1 being more abundant in the medullar region than in the cortex. The melano-macrophage centres found in the interstitial compartment displayed glycans terminating with Galbeta1,3GalNAc. The basal lamina separating the germinal and interstitial compartments exhibited glycans with terminal/internal mannose, internal betaGlcNAc, and terminal Neu5Acalpha2,6Gal/GalNAc, and Neu5AcGalbeta1,3GalNAc, Galbeta1,3GalNAc (PNA), Galbeta1,4GlcNAc, GalNAc, alphaGal, and alphaL-Fuc. In the germinal compartment, the Sertoli cells expressed only glycans terminating with Neu5Acalpha2,3Galbeta1,4GlcNAc in the apical and supra-nuclear lateral surface of the spermatonial cysts located in the distal part of the seminiferous lobules. Primary spermatocytes exhibited oligosaccharides terminating with Galbeta1,3GalNAc and alphaGalNAc in the cytoplasm and nucleus, respectively. The spermatids contained highly mannosylated glycans terminating with GalNac, alphaGal, and alphaL-Fuc. The head of spermatozoa expressed a more complex glycosylation pattern characterized by the additional presence of oligosaccharides terminating with Neu5Acalpha2,3Galbeta1,4GlcNAc, Neu5AcGalbeta1,3GalNAc, Neu5AcGalNAcalpha1,3(LFuca1,2)Galbeta1,3/4GlcNAcbeta1, GalNAcalpha1,3(LFucalpha1,2)Galbeta1,3/4GlcNAcbeta1. The comparison with previous lectin histochemical studies carried out in other fish species reveals a specific glycosylation pattern of Senegalese sole testicular structures and spermatozoa head.

Equine mandibular gland: in situ characterisation of sialoderivatives

Sialic acids modulate the metabolite transport across membranes and may be involved in protection against pathogenic agents. The presence of sialoderivatives in the equine mandibular gland requires further study. To biochemically visualise in situ the presence of sialoderivatives, by means of mild and strong periodate oxidation and alcoholic saponification, combined with lectin histochemistry and sialidase digestion in order to hypothesise roles for detached sialoderivatives. Mandibular glands were removed from 8 mature horses of both sexes and subjected to histochemical procedures, including periodate oxidation, saponfication and lectin staining. Controls were based upon the omission of peroxidase-conjugated lectins and respective enzyme-free buffers. The reactivities of PNA and RCA I lectins were affected by sialidase treatment, whether preceded by saponification or not, showing that the dimer N-acetyl-sialic acid-beta-Gal was linked (1-3)GalNAc and (1-4)GlcNAc. In acinar cells the sequence sialic acid-beta-Gal(1-3)GalNAc showed sialic residues acetylated at C4 only and at C4 and C7 and/or C8 and/or C9(alpha2-6Gal) in both sexes, while in female mandibular gland also C4 and C9(alpha2-3Gal) acetylated residues were present. Sialic acid linked to beta-Gal(1-4)GlcNAc was prevalently C4 and C7 and/or C8 and/or C9(alpha2-6Gal and alpha2-3Gal) acetylated, whereas only a minor quantity showed acetyl groups at C7 and/or C8 and/or C9(alpha2-6Gal) in the acinar cells of both sexes. The great variety of sialic acid residues expressed by equine mandibular gland could assume an important role in the defensive mechanisms towards pathogen agents and, compared with those of cattle, probably represents an example of molecular species-specificity related to different alimentary habits.

Ultrastructure and lectin cytochemistry of secretory cells in lingual glands of the Japanese quail (Coturnix coturnix japonica).

In the present study, as continuation of our previous research, Japanese quail (Coturnix coturnix japonica) lingual glands were investigated by means of transmission electron microscopy (TEM) to understand the cytoarchitecture and the subcellular sugar distribution within the different secretory structures. Indeed, glycosidic residues were visualized by applying an indirect technique of binding and the terminal sialoglycoconjugate sequences were characterized by employing sialidase digestion combined with lectin affinity. The ultrastructural analysis revealed an unusual cytoarchitecture of the caudal portion of anterior lingual gland that was composed of both secretory cells, filled with granules, and non-secretory cells, filled with mitochondria. Conversely, the posterior lingual gland was composed of secretory units of lingual glands only containing mucous cells filled with secretory granules with a variable morphology, including bipartite features characterized by an electron-lucent matrix and one or more electron-dense areas. Actual findings further supported that the quail lingual glands produce sialoglycoconjugates characterized by a heterogeneous composition. In conclusion, the cytological characteristics and the carbohydrate composition of quail lingual glands suggest that, analogously to mammal salivary glands, avian lingual glands could also be involved in several functions that can be correlated with the occurrence of sialic acids.

Immunogold study on lectin binding in the porcine zona pellucida and granulosa cells

An ultrastructural localization of lectin receptors on the zona pellucida (ZP) of porcine antral oocytes and on the granulosa cells was performed using a panel of horseradish peroxidase-labelled lectins in conjunction with antiperoxidase antibody and protein A-gold. In some cases, lectin incubation was preceded by sialidase digestion. WGA-, Con-A-, UEA-I-, RCA-I-, PNA- and SBA-reactive sites were distributed differently in the porcine ZP. Sialidase digestion increased the positivity obtained with RCA-I and it was necessary to promote PNA and SBA reactivity. These results indicated that the ZP contained N-acetylglucosamine, a-mannose, a-fucose, b-Gal-(1-4)GlcNAc, b-Gal- (1-3)GalNAc, b-GalNAc and sialic acid residues. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the ZP, thus suggesting that the oocyte and granulosa cells are the site of synthesis of ZP glucidic determinants.

The Ductus Epididymis of the Alpaca: Immunohistochemical and Lectin Histochemical Study

Our objective was to characterize epithelial cells lining the epididymal duct (caput, corpus, cauda) of the alpaca using AE1/AE3 cytokeratin antibodies and a battery of different lectins: Con-A, UEA-I, LTA WGA, GSA-II, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA. Sialidase digestion and deglycosylation pre-treatments were also employed. The principal cells (PCs) along the epididymis showed differences in immunostaining patterns toward keratin antibodies. Lectin histochemistry demonstrated variations in the content and distribution of glycosidic residues of glycoconjugates in different epididymal regions. In particular, staining of the Golgi zone in the epithelial PCs was interpreted as evidence for synthesis and secretion of O- and N-linked oligosaccharides. In the caput, the apical mitochondria-rich cells contained mainly beta-GalNAc, subterminal alpha-GalNAc, alpha-Gal and Neu5Ac alpha2,3Gal residues. Conversely, in the corpus they were particularly rich in alpha-GalNac and beta-Gal-(1-3)-d-GalNAc linked to sialic acid moieties. Basal cells mainly expressed beta-GalNAc and alpha-Gal in the caput, alpha-Gal in the corpus and alpha-Fuc and beta-GalNAc in the cauda. The differences in immunostaining patterns and in lectin histochemistry in the alpaca epididymis reported in this investigation seem to be related to regional differences in function.

Glycoprofile of the different cell types present in the mucosa of the horse guttural pouches

Histochemical characterization of the equine guttural pouches was performed using lectins combined with sialidase digestion and deglycosylation pre-treatments. The goblet cells contained O- and N-linked oligosaccharides with α-Fuc, GlcNAc moieties whereas β-GalNAc, β-Gal-(1–3)-GalNAc, β-Gal-(1–4)-GlcNAc and α-Gal residues belonged only to O-linked glycoproteins. The acinar and ductal cells expressed α-Man/α-Glc in N-linked oligosaccharides, GlcNAc in both O- and N-glycoproteins and β-GalNAc, β-Gal-(1–3)-GalNAc, β-Gal-(1–4)-GlcNAc and α-Gal residues included in O-linked glycoproteins. The Golgi area of the epithelial lining expressed α-Fuc in O-linked glycoproteins, internal GlcNAc in N-linked glycoproteins and large amounts of sialic acid residues linked to subterminal β-GalNAc, Galβ1,4GlcNAc and Galβ1,3GalNAc. High amounts of sulpho-carbohydrates and of sialic acids (α2,3–6), linked to-α/β-Gal and sialic acids (α2–6) linked to β-GalNAc, were also demonstrated. Such diversity of the mucin saccharide residues may be implicated in the binding of macromolecules such as those of bacterial or viral etiology, thus playing a role in the organism's host-defense mechanism in the guttural pouches.

Immunohistochemical and lectin histochemical analysis of the alpaca efferent ducts.

An immunohistochemical and lectin histochemical study of the efferent ducts was performed in the alpaca. Two types of epithelium, consisting of principal and ciliated cells, were detected on the basis of the different cytokeratins expression and lectin binding pattern. AE1/AE3 and 13 cytokeratin antibodies intensely immunostained the entire cytoplasm of type I PCs, whereas AE1/AE3, but not anti cytokeratin 13, immunoreacted in type II principal cells along the apical, lateral and basal plasma-membrane. The histochemical characterization of the epithelial cells was carried out using a battery of different lectins: Con-A, UEA-I, LTA, WGA, GSA-II, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA. Sialidase digestion and deglycosilation pre-treatments were also employed. In type I principal cells, the staining of the Golgi zone was interpreted giving evidence for the synthesis and secretion of O- and N-linked oligosaccharides. In particular, alpha-Man/alpha-Glc, GlcNAc, beta-Gal-(1-4)-GlcNAc, Neu5Acalpha2,3Gal and Neu5Acalpha2,6Gal/GalNAc residues were included in both O- and N-linked glycans, whereas alpha-Fuc, beta-GalNAc and alpha-Gal were only found in O-linked oligosaccharides; alpha-GalNac and beta-Gal-(1-3)-D-GalNAc were found subterminal to sialic acid moieties and they were included in O- and N-glycans. In type II principal cells, the lectin staining was observed in the apical cytoplasmic granules and in vacuoles that were interpreted as components of an elaborate endocytotic apparatus specialized for the uptake of particulate material and fluid from the lumen. These results suggest the existence of two structurally different epithelial segments along the ductuli efferentes of the alpaca, with a high degree of compartmentalization of the secretory and absorptive activities.

Variability, Heritability and Environmental Determinants of Human Plasma N-Glycome

Plasma glycans were analyzed in 1008 individuals to evaluate variability and heritability, as well as the main environmental determinants that affect glycan structures. By combining HPLC analysis of fluorescently labeled glycans with sialidase digestion, glycans were separated into 33 chromatographic peaks and quantified. A high level of variability was observed with the median ratio of minimal to maximal values of 6.17 and significant age- and gender-specific differences. Heritability estimates for individual glycans varied widely, ranging from very low to very high. Glycome-wide environmental determinants were also detected with statistically significant effects of different variables including diet, smoking and cholesterol levels.

Glycocomposition of the apocrine interdigital gland secretions in the fallow deer ( Dama dama)

The secretions of the tubular interdigital glands were investigated by conventional (Periodic-Acid Schiff, Alcian-Blue at different pH, Low Iron Diamine and High Iron Diamine) and lectin (Con-A, UEA-I, LTA, WGA, GSA-II, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA) histochemical methods in adult males and females of different age of fallow deer during the breeding season. Sialidase digestion and deacetylation pre-treatment were also employed in conjunction with lectin histochemistry. The glandular epithelium consisted of a single layer of low columnar cells with typical apical protrusions. No substantial differences of the above histochemical staining in relation to sex and age were observed. Conventional histochemical staining revealed that the interdigital glands secreted neutral glycoproteins whereas acidic glycocomponents did not seem to be present. Lectin histochemical technique allowed us to disclose a great heterogeneity of glycoproteins with N- and O-linked oligosaccharides containing α- d-Man/α- d-Glc, GlcNAc, α-Fuc, terminal β- d-Gal-(1-3)- d-GalNAc, - d-Gal-(1-4)- d-GlcNAc, α-Gal and β-GalNAc residues. β-GalNAc and disaccharide β- d-Gal-(1-3)- d-GalNAc were also found as subterminal to sialyl moieties. The lack of sexual and age-related differences in the glucidic content of the glandular secretions seems to indicate that the glycoderivatives may play only an accessory role in the production of odoriferous signals in fallow deer.

Localization of influenza virus sialoreceptors in equine respiratory tract.

This study was performed to identify the equine respiratory tract areas which express the specific receptor for equine influenza virus; findings may be useful to provide new ways to treat the infectious disease. The present work aims to visualize in situ the presence of sialoderivatives in the horse respiratory tract in order to localize sialoderivatives acting as influenza virus receptors. To this purpose, nasal mucosae, trachea, bronchus and lung parenchyma were removed from 8 mature horses of both sexes. We performed sialic acid characterization by means of mild and strong periodate oxidation and saponification, combined with lectin histochemistry and sialidase digestion, in addition to the direct evidentiation of sialic acid residues. No differences were shown between sexes. Sialic acid residues are present in the nasal mucous cell secretion, where they are linked to galactose by means of alpha2-3 linkage and are mainly C9 acetylated, and in the nasal and tracheal epithelial lining, where they are represented by periodate labile residues (alpha2-3)- and/or (alpha2-6)- linked to galactose. Specific receptors for equine influenza viruses are present at the nasal and tracheal epithelial lining cell coat levels, and in some trachea epithelial cells, but the horse possesses a preventive defence, which consists of the secretion of a mucous layer at nasal level, which could specifically inactivate the hemagglutinins of equine influenza virus; in addition, it expresses other sialoreceptors which can mask the influenza specific ones.

Lectin histochemistry for in situ profiling of rat colon sialoglycoconjugates.

The growing interest in glycoconjugates expressed and released by the epithelium of the intestinal mucosa is tightly related to the multiple functional roles attributed to sialic acid and its derivatives. In the present work, biotin and HRP conjugated lectins were used to detect the sialylation pattern and to identify specific structural features of sialoderivatives in the rat colon. In particular, the occurrence and distribution of sialic acids linked alpha2,6 to D-Gal/D-GalNAc and alpha2,3 to D-Gal were directly demonstrated with SNA and MAL II binding, respectively. In addition, in order to by-pass the specificity problems of SNA and MAL II as histochemical reagents, as well as to look for additional and complementary information about acetylation degree and sites, we combined sialidase digestion, potassium hydroxide deacetylation, and differential periodate oxidation with PNA and DBA binding. The data showed the distribution and structure of sialic acid-beta-D-Gal(1-3)-D-GalNAc and sialic acid-D-GalNac sequences, which proved to be widely distributed as cellular components or secretory products in surface goblet cells and crypt cells of the colonic epithelium. A high degree of O-acetylation, with acetyl groups mainly at 9 and 4 positions, was found, showing an increasing gradient from the proximal to distal portion of the colon. These results, which largely reproduce the sialylation pattern in other species, contribute new insights in defining the tissue specific expression of sialoderivatives in the colonic mucosa, and testify to their high heterogeneity which the wide range of sialic acid functional correlates in the intestinal tract depend on.

Characterization of glycoconjugates in the secretory epithelium of the equine ampulla ductus deferentis.

The present work was undertaken to determine the glycoconjugates secreted by the epithelium of the equine ampulla ductus deferentis, using conventional (PAS, AB pH 2.5, AB pH 1.0) and lectin histochemical procedures in conjunction with enzymatic digestion and chemical treatment. The presence of abundant apical cytoplasmic blebs suggests that the equine ampulla secretes its products mainly in an apocrine manner. Glandular cells secrete neutral and acidic sialylated glycoconjugates as revealed by conventional histochemical procedures. Lectin histochemistry helped us to discover the following histological positive sites: the mucosal cells, the glandular epithelial cells, the apical cytoplasmic blebs and the basal cells. The ampullary secretions contained both glycoproteic material (revealed by Con-A-, LCA-, GSA-II-, WGA-, RCA-I- positivity) and sialomucins (evidenced by the reactivity of GSA-II, SBA, PNA and RCA-I after sialidase digestion) having different functional roles. The mucosal cells reacted with Con-A, LCA, and also with sialidase/GSA-II-, SBA-, PNA-, and RCA-I sequences, contributing to the chemical heterogeneity of ampullary secretions. DBA lectin was a specific marker for basal cells. The results obtained were compared with our previous findings regarding the differences in the lectin binding pattern of the plasma-membrane of equine sperm collected from epididymal cauda and the ampulla ductis deferentis. Our results support other studies that indicate that ampullary secretions are involved in altering the plasma-membrane glycoconjugates of spermatozoa, contributing to their maturation.

Development of Immunoglobulin A Nephropathy- Like Disease in β-1,4-Galactosyltransferase-I-Deficient Mice

Beta4 galactosylation of glycoproteins plays important roles in protein conformation, stability, transport, and clearance from the circulation. Recent studies have revealed that aberrant glycosylation causes various human diseases. Here we report that mice lacking beta-1,4-galactosyltransferase (beta4GalT)-I, which transfers galactose to the terminal N-acetylglucosamine of N- and O-linked glycans in a beta-1,4 linkage, spontaneously developed human immunoglobulin A nephropathy (IgAN)-like glomerular lesions with IgA deposition and expanded mesangial matrix. beta4GalT-I-deficient mice also showed high serum IgA levels with increased polymeric forms as in human IgAN. IgAN is the most common form of glomerulonephritis, and a significant proportion of patients progress to renal failure. However, pathological molecular mechanisms of IgAN are poorly understood. In humans, abnormal character of serum IgA, especially serum IgA1 with aberrant galactosylation and sialylation of O-glycans in its hinge region is thought to contribute to the pathogenesis of IgAN. Mouse IgA has N-glycans but not O-glycans, and beta4-galactosylation and sialylation of the N-glycans on the serum IgA from beta4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We propose that carbohydrates of serum IgA are involved in the development of IgAN, whether the carbohydrates are O-glycans or N-glycans.

Evaluation of laser microdissection as a tool in cancer glycomic studies

Laser microdissection (LMD) is a recent development that enables the isolation of specific cell populations from tissue sections. This study focuses on the potential of LMD as a tool in cancer glycomics using colon cancer as a model. LMD was performed on hematoxylin and eosin stained frozen tissue sections. Tumor cells and normal epithelial cells were selectively microdissected. N-Glycans from the LMD- and the bulk tissue-derived samples were liberated by hydrazinolysis and then labeled with 2-aminopyridine. After sialidase digestion, the resulting asialo- N-glycans were analyzed by normal and reversed phase HPLC combined with mass spectrometry. Comparison of the various N-glycan profiles with the aid of LMD identified seven characteristic N-glycans with significantly different expression profiles between normal and cancerous cells that could not be detected by conventional analysis. Thus, LMD is a potent and useful tool for analyzing variations in the expression of N-glycans by overcoming the problem of tissue sample heterogeneity.

Histochemical analysis of glycoconjugates in the domestic cat testis.

The localization and characterization of oligosaccharide sequences in the cat testis was investigated using 12 lectins in combination with the beta-elimination reaction, N-Glycosidase F and sialidase digestion. Leydig cells expressed O-linked glycans with terminal alphaGalNAc (HPA reactivity) and N-glycans with terminal/internal alphaMan (Con A affinity). The basement membrane showed terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,3GalNAc, alpha/betaGalNAc, and GlcNAc (SNA, PNA, HPA, SBA, GSA II reactivity) in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc (RCA120 staining) and alphaMan in N-linked oligosaccharides; in addition, terminal Neu5acalpha2,3Galbeta1,4GlcNac, Forssman pentasaccharide, alphaGal, alphaL-Fuc and internal GlcNAc (MAL II, DBA, GSA I-B4, UEA I, KOH-sialidase-WGA affinity) formed both O- and N-linked oligosaccharides. The Sertoli cells cytoplasm contained terminal Neu5Ac-Galbeta1,4GlcNAc, Neu5Ac-betaGalNAc as well as internal GlcNAc in O-linked glycans, alphaMan in N-linked glycoproteins and terminal Neu5Acalpha2,6Gal/ GalNAc in both O- and N-linked oligosaccharides. Spermatogonia exhibited cytoplasmic N-linked glycoproteins with alphaMan residues. The spermatocytes cytoplasm expressed terminal Neu5Acalpha2,3Galbeta1,4 GlcNAc and Galbeta1,3GalNAc in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-linked glycoconjugates. The Golgi region showed terminal Neu5Acalpha2,3Galbeta1,4GlcNac, Galbeta1,4GlcNAc, Forssman pentasaccharide, and alphaGalNAc in O-linked oligosaccharides, alphaMan and terminal betaGal in N-linked oligosaccharides. The acrosomes of Golgi-phase spermatids expressed terminal Galbeta1,3GalNAc, Galbeta1,4GlcNAc, Forssmann pentasaccharide, alpha/betaGalNAc, alphaGal and internal GlcNAc in O-linked oligosaccharides, terminal alpha/betaGalNAc, alphaGal and terminal/internal alphaMan in N-linked glycoproteins. The acrosomes of cap-phase spermatids lacked internal Forssman pentasaccharide and alphaGal, while having increased alpha/betaGalNAc. The acrosomes of elongated spermatids did not show terminal Galbeta1,3GalNAc, displayed terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-glycans and Neu5Ac-Galbeta1,3GalNAc in O-linked oligosaccharides.