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Background: 25-Hydroxyvitamin D (25(OH)D) deficiency is the most common complication of kidney disease. Previous studies have suggested that 25(OH)D deficiency is involved in the pathogenesis of anemia in kidney disease subjects not requiring dialysis. However, these associations have not been investigated in peritoneal dialysis (PD) patients. Objectives: The aim of this study was to elucidate the prospective relationship between 25-Hydroxyvitamin D insufficiency and anemia in PD Patients.Materials and Methods: In a cross-sectional study, 80 PD participants were included. Participants were divided into two groups based on baseline 25(OH)D3 concentrations: group 1, 25(OH)D3 levels

Congenital disorders of glycosylation (CDGs) are caused by defects in genes that participate in biosynthetic glycosylation pathways. To date, 19 different genetic defects in N-glycosylation, 17 in O-glycosylation, and 21 in multiple glycosylation are known. Current diagnostic testing of CDGs largely relies on indirect analysis of glycosylation of serum transferrin. Such analysis alone is insufficient to diagnose many of the known glycosylation disorders. To improve the diagnosis of these groups of CDGs, we have developed serum or plasma N- and O-glycan profiling using a combination of MALDI-TOF/MS and LC-MS/MS technologies. Using this approach, we analyzed samples from nine patients with different known multiple glycosylation disorders, including three with COG deficiencies, one with TMEM165-CDG, two with PGM1-CDG, and three with SLC35A2-CDG, and one patient with combined type I and type II of unknown molecular etiology. Measurement of the relative quantities of various N- and O-glycan species clearly differentiates patients and controls. Our study demonstrates that structural analysis and quantitation of combined N- and O-glycan profiles are reliable diagnostic tools for CDGs.

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.

Loss of T-synthase (uridine diphosphate galactose:N-acetylgalactosaminyl-α1-Ser/Thr β3galactosyltransferase), a key enzyme required for the formation of mucin-type core 1 O-glycans, is observed in several human diseases, including cancer, Tn syndrome and IgA nephropathy, but current methods to assay the enzyme use radioactive substrates and complicated isolation of the product. Here we report the development of a novel fluorescent assay to measure its activity in a variety of tumor cell lines. Deficiencies in T-synthase activity correlate with mutations in the gene encoding the molecular chaperone Cosmc that is required for folding the T-synthase. This new high-throughput assay allows for facile screening of tumor specimens and other biological material for T-synthase activity and could be used diagnostically.

The expression of ABO(H) blood group antigens causes deletion of cells that generate self-specific antibodies to these antigens but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against such pathogens, given these limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectin-4 (Gal-4) and Gal-8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing Escherichia coli while failing to alter the viability of other E. coli strains or other Gram-negative or Gram-positive organisms both in vitro and in vivo. The killing activity of both Gal-4 and Gal-8 is mediated by their C-terminal domains, occurs rapidly and independently of complement and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that express blood group-like antigens on their surface.

Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1-derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn(-/-) mice). EHC T-syn(-/-) mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn(-/-) mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn(-/-) lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn(-/-) defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn(-/-) mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn(-/-) pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.

Neoplastic lesions typically express specific carbohydrate antigens on glycolipids, mucins, and other glycoproteins. Such antigens are often under epigenetic control and are subject to reversion and loss upon therapeutic selective pressure. We report here that two of the most common tumor-associated carbohydrate antigens, Tn and sialyl Tn (STn), result from somatic mutations in the gene Cosmc that encodes a molecular chaperone required for formation of the active T-synthase. Diverse neoplastic lesions, including colon cancer and melanoma-derived cells lines, expressed both Tn and STn antigen due to loss-of-function mutations in Cosmc. In addition, two human cervical cancer specimens that showed expression of the Tn/STn antigens were also found to have mutations in Cosmc and loss of heterozygosity for the cross-linked Cosmc locus. This is the first example of somatic mutations in multiple types of cancers that cause global alterations in cell surface carbohydrate antigen expression. [Cancer Res 2008;68(6):1636–46]

A glycan microarray was developed by using 2,6-diaminopyridine (DAP) as a fluorescent linker and printing of the glycan-DAP conjugates (GDAPs) on epoxy-activated glass slides. Importantly, all coupled GDAPs showed a detectable level of concentration-dependent GDAP fluorescence under blue laser excitation (495 nm) that can be used for both grid location and on-slide quantification. A glycan array including a large number of GDAP's derived from natural and commercially available free glycans was constructed and glycan interactions with various plant lectins were investigated. In addition, binding parameters of lectins to glycans were obtained by varying both the amount of GDAPs on the array and the lectin concentration in analyses. These data demonstrate the general utility of GDAP microarrays for functional glycomic analyses and for determining binding parameters of glycan binding proteins (GBPs).

Altered intestinal O-glycan expression has been observed in patients with ulcerative colitis and colorectal cancer, but the role of this alteration in the etiology of these diseases is unknown. O-glycans in mucin core proteins are the predominant components of the intestinal mucus, which comprises part of the intestinal mucosal barrier. Core 3–derived O-glycans, which are one of the major types of O-glycans, are primarily expressed in the colon. To investigate the biological function of core 3–derived O-glycans, we engineered mice lacking core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT), an enzyme predicted to be important in the synthesis of core 3–derived O-glycans. Disruption of the C3GnT gene eliminated core 3–derived O-glycans. C3GnT-deficient mice displayed a discrete, colon-specific reduction in Muc2 protein and increased permeability of the intestinal barrier. Moreover, these mice were highly susceptible to experimental triggers of colitis and colorectal adenocarcinoma. These data reveal a requirement for core 3–derived O-glycans in resistance to colonic disease.

BACKGROUND Primary deficiencies in mannosylation of N-glycans are seen in a majority of patients with congenital disorders of glycosylation (CDG). We report the discovery of a series of novel N-glycans in sera, plasma, and cultured skin fibroblasts from patients with CDG having deficient mannosylation. METHOD We used LC-MS/MS and MALDI-TOF-MS analysis to identify and quantify a novel N-linked tetrasaccharide linked to the protein core, an N-tetrasaccharide (Neu5Acα2,6Galβ1,4-GlcNAcβ1,4GlcNAc) in plasma, serum glycoproteins, and a fibroblast lysate from patients with CDG caused by ALG1 [ALG1 (asparagine-linked glycosylation protein 1), chitobiosyldiphosphodolichol β-mannosyltransferase], PMM2 (phosphomannomutase 2), and MPI (mannose phosphate isomerase). RESULTS Glycoproteins in sera, plasma, or cell lysate from ALG1-CDG, PMM2-CDG, and MPI-CDG patients had substantially more N-tetrasaccharide than unaffected controls. We observed a >80% decline in relative concentrations of the N-tetrasaccharide in MPI-CDG plasma after mannose therapy in 1 patient and in ALG1-CDG fibroblasts in vitro supplemented with mannose. CONCLUSIONS This novel N-tetrasaccharide could serve as a diagnostic marker of ALG1-, PMM2-, or MPI-CDG for screening of these 3 common CDG subtypes that comprise >70% of CDG type I patients. Its quantification by LC-MS/MS may be useful for monitoring therapeutic efficacy of mannose. The discovery of these small N-glycans also indicates the presence of an alternative pathway in N-glycosylation not recognized previously, but its biological significance remains to be studied.

Pseudomonas aeruginosa infection of patients with cystic fibrosis (CF) is a leading cause of their morbidity and mortality. Pathogenesis is initiated in part by molecular interactions of P. aeruginosa with carbohydrate residues in airway mucins that accumulate in the lungs of patients with this disease. To explore the nature of the glycans recognized by a stable, mucoid, alginate-producing strain P. aeruginosa 8830 we generated a genetically modified Pa8830 expressing green fluorescent protein (Pa3380-GFP). We tested its binding to a panel of glycolipids and neoglycolipids in which selected glycans were covalently attached to dipalmitoyl phosphatidylethanolamine and analyzed on silica gel surfaces. Among all glycans tested, Pa8830-GFP bound best to sialyl-Le(x)-containing glycan NeuAc(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc-R and bound weakly to H-type blood group Fucalpha1-2Galbeta1-4GlcNAc-R, sialyl-lactose, and Le(x), and exhibited little binding toward non-fucosylated derivatives. Interestingly, while Pa8830-GFP bound to the glycosphingolipid asialoGM1, it did not appear to bind to a wide variety of other glycosphingolipids including GM1, GM2, asialoGM2, and sulfatide. These results indicate that P. aeruginosa 8830 has preferential binding to sialyl-Le(x)-containing glycans and has weak recognition of related fucose- and sialic acid-containing glycans. The finding that Pa8830 binds sialyl-Le(x)-containing glycans, which occur at increased levels in mucins from CF patients, is consistent with studies of other strains of P. aeruginosa and further suggests that such glycans on CF mucins contribute to disease pathogenesis.

The new field of functional glycomics encompasses information about both glycan structure and recognition by carbohydrate-binding proteins (CBPs) and is now being explored through glycan array technology. Glycan array construction, however, is limited by the complexity of efficiently generating derivatives of free, reducing glycans with primary amines for conjugation. Here we describe a straightforward method to derivatize glycans with 2,6-diaminopyridine (DAP) to generate fluorescently labeled glycans (glycan-DAP conjugates or GDAPs) that contain a primary amine for further conjugation. We converted a wide variety of glycans, including milk sugars, N-glycans, glycosaminoglycans and chitin-derived glycans, to GDAPs, as verified by HPLC and mass spectrometry. We covalently conjugated GDAPs to N-hydroxysuccinimide (NHS)-activated glass slides, maleimide-activated protein, carboxylated microspheres and NHS-biotin to provide quantifiable fluorescent derivatives. All types of conjugated glycans were well-recognized by appropriate CBPs. Thus, GDAP derivatives provide versatile new tools for biologists to quantify and covalently capture minute quantities of glycans for exploring their structures and functions and generating new glycan arrays from naturally occurring glycans.

Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed glycan reductive isotope labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [(12)C(6)]aniline and [(13)C(6)]aniline. These dual-labeled aniline-tagged glycans can be recovered by reverse-phase chromatography and can be quantified based on ultraviolet (UV) absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins, using this method. This technique allows linear relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of glycomics.

Compromised epithelial cell integrity is a common feature associated with chronic lung inflammatory states such as asthma. While epithelial cell damage is largely due to sustained effects of inflammatory mediators localized to airways, the subsequent process of epithelial cell differentiation is attributed to members of the transmembrane receptor tyrosine kinase family called the ErbB's. MUC4, a large molecular weight membrane-bound glycoprotein, has recently been identified as a potential ligand for the ErbB-2 receptor. In this study, we investigated the possible role of interleukin-9 (IL-9), a Th2 cytokine, on MUC4 expression using a lung cancer cell line, NCI-H650. We determined that IL-9 up-regulates MUC4 expression in a time and concentration-dependent fashion. Nuclear run-on assays indicated transcriptional regulation of MUC4 while no post-transcriptional mRNA stabilization was observed by actinomycin D chase experiments. IL-9 also increased MUC4 glycoprotein expression as determined by Western blots using a monoclonal antibody specific for a non-tandem repeat region on ASGP-2 region of MUC4. Furthermore, a JAK3-selective inhibitor 4-(4′-hydroxyphenyl) amino-6, 7-dimethoxyquinazoline (WHI-P131), substantially reduced IL-9-induced MUC4 mRNA expression in a dose-dependent fashion. These results implicate a potential role for IL-9 upon MUC4 expression in human airway epithelial cells.

Cystic fibrosis (CF) is the most lethal genetic disorder in Caucasians and is characterized by the production of excessive amounts of viscous mucus secretions in the airways of patients, leading to airway obstruction, chronic bacterial infections, and respiratory failure. Previous studies indicate that CF-derived airway mucins are glycosylated and sulfated differently compared with mucins from nondiseased (ND) individuals. To address unresolved questions about mucin glycosylation and sulfation, we examined O-glycan structures in mucins purified from mucus secretions of two CF donors versus two ND donors. All mucins contained galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), and sialic acid (Neu5Ac). However, CF mucins had higher sugar content and more O-glycans compared with ND mucins. Both ND and CF mucins contained GlcNAc-6-sulfate (GlcNAc-6-Sul), Gal-6-Sul, and Gal-3-Sul, but CF mucins had higher amounts of the 6-sulfated species. O-glycans were released from CF and ND mucins and derivatized with 2-aminobenzamide (2-AB), separated by ion exchange chromatography, and quantified by fluorescence. There was nearly a two-fold increase in sulfation and sialylation in CF compared with ND mucin. High performance liquid chromatography (HPLC) profiles of glycans showed differences between the two CF samples compared with the two ND samples. Glycan compositions were defined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Unexpectedly, 260 compositional types of O-glycans were identified, and CF mucins contained a higher proportion of sialylated and sulfated O-glycans compared with ND mucins. These profound structural differences in mucin glycosylation in CF patients may contribute to inflammatory responses and increased pathogenesis by Pseudomonas aeruginosa.

SummaryGalectin-1 (Gal-1) and galectin-3 (Gal-3) are widely expressed galectins with immunoregulatory functions in animals. To explore their glycan specificity, we developed microarrays of naturally occurring glycans using a bifunctional fluorescent linker, 2-amino-N-(2-aminoethyl)-benzamide (AEAB), directly conjugated through its arylamine group by reductive amination to free glycans to form glycan-AEABs (GAEABs). Glycans from natural sources were used to prepare over 200 GAEABs, which were purified by multidimensional high-pressure liquid chromatography and covalently immobilized onto N-hydroxysuccinimide-activated glass slides via their free alkylamine. Fluorescence-based screening demonstrated that Gal-1 recognizes a wide variety of complex N-glycans, whereas Gal-3 primarily recognizes poly-N-acetyllactosamine-containing glycans independent of N-glycan presentation. GAEABs provide a general solution to glycan microarray preparation from natural sources for defining the specificity of glycan-binding proteins.

BackgroundRecent studies have identified MUC4 mucin as a ligand for activation of ErbB2, a receptor tyrosine kinase that modulates epithelial cell proliferation following epithelial damage in airways of asthmatics. In this study, we investigated the potential role of IL-4, one of the Th2 inflammatory cytokines persistent in asthmatic airways, in regulating MUC4 expression using a cell line NCI-H650.MethodsReal time PCR analysis was performed to determine concentration and time dependent effects of IL-4 uponMUC4expression. Nuclear run on experiments were carried out to explore potential transcriptional modulation. Western blotting experiments using a monoclonal antibody specific to ASGP-2 domain of MUC4 were performed to analyze MUC4 glycoprotein levels in plasma membrane fractions. To analyze potential signal transduction cascades, IL-4 treated confluent cultures were co-incubated, separately with a pan-JAK inhibitor, a JAK-3 selective inhibitor or a MEK-1, 2 (MAPK) inhibitor at various concentrations beforeMUC4transcript analysis. Corresponding transcription factor activation was tested by western blotting using a monoclonal p-STAT-6 antibody.ResultsMUC4levels increased in a concentration and time specific fashion reaching peak expression at 2.5 ng/ml and 8 h. Nuclear run on experiments revealed transcriptional enhancement. Corresponding increases in MUC4 glycoprotein levels were observed in plasma membrane fractions. Pan-JAK inhibitor revealed marked reduction in IL-4 stimulatedMUC4levels and JAK3 selective inhibitor down-regulated MUC4 mRNA expression in a concentration-dependent fashion. In accordance with the above observations, STAT-6 activation was detected within 5 minutes of IL-4 stimulus. No effect inMUC4levels was observed on using a MAPK inhibitor.ConclusionThese observations signify a potential role for IL-4 in MUC4 up-regulation in airway epithelia.