Effects of Size and Geographical Origin on Atlantic salmon, Salmo salar, Mucin O-Glycan Repertoire.

Diseases cause ethical concerns and economic losses in the Salmonid industry. The mucus layer comprised of highly O-glycosylated mucins is the first contact between pathogens and fish. Mucin glycans govern pathogen adhesion, growth and virulence. The Atlantic salmon O-glycome from a single location has been characterized and the interindividual variation was low. Because interindividual variation is considered a population-based defense, hindering the entire population from being wiped out by a single infection, low interindividual variation among Atlantic salmon may be a concern. Here, we analyzed the O-glycome of 25 Atlantic salmon from six cohorts grown under various conditions from Sweden, Norway and Australia (Tasmania) using mass spectrometry. This expanded the known Atlantic salmon O-glycome by 60% to 169 identified structures. The mucin O-glycosylation was relatively stable over time within a geographical region, but the size of the fish affected skin mucin glycosylation. The skin mucin glycan repertoires from Swedish and Norwegian Atlantic salmon populations were closely related compared with Tasmanian ones, regardless of size and salinity, with differences in glycan size and composition. The internal mucin glycan repertoire also clustered based on geographical origin and into pyloric cecal and distal intestinal groups, regardless of cohort and fish size. Fucosylated structures were more abundant in Tasmanian pyloric caeca and distal intestine mucins compared with Swedish ones. Overall, Tasmanian Atlantic salmon mucins have more O-glycan structures in skin but less in the gastrointestinal tract compared with Swedish fish. Low interindividual variation was confirmed within each cohort. The results can serve as a library for identifying structures of importance for host-pathogen interactions, understanding population differences of salmon mucin glycosylation in resistance to diseases and during breeding and selection of strains. The results could make it possible to predict potential vulnerabilities to diseases and suggest that inter-region breeding may increase the glycan diversity.

Stress Impairs Skin Barrier Function and Induces α2-3 Linked N-Acetylneuraminic Acid and Core 1 O-Glycans on Skin Mucins in Atlantic Salmon, Salmo salar.

The skin barrier consists of mucus, primarily comprising highly glycosylated mucins, and the epithelium. Host mucin glycosylation governs interactions with pathogens and stress is associated with impaired epithelial barrier function. We characterized Atlantic salmon skin barrier function during chronic stress (high density) and mucin O-glycosylation changes in response to acute and chronic stress. Fish held at low (LD: 14-30 kg/m3) and high densities (HD: 50-80 kg/m3) were subjected to acute stress 24 h before sampling at 17 and 21 weeks after start of the experiment. Blood parameters indicated primary and secondary stress responses at both sampling points. At the second sampling, skin barrier function towards molecules was reduced in the HD compared to the LD group (Papp mannitol; p < 0.01). Liquid chromatography-mass spectrometry revealed 81 O-glycan structures from the skin. Fish subjected to both chronic and acute stress had an increased proportion of large O-glycan structures. Overall, four of the O-glycan changes have potential as indicators of stress, especially for the combined chronic and acute stress. Stress thus impairs skin barrier function and induces glycosylation changes, which have potential to both affect interactions with pathogens and serve as stress indicators.

Exploring the Arctic Charr Intestinal Glycome: Evidence of Increased N-Glycolylneuraminic Acid Levels and Changed Host-Pathogen Interactions in Response to Inflammation.

Disease outbreaks are a limiting factor for the sustainable development of the aquaculture industry. The intestinal tract is covered by a mucus layer mainly comprised by highly glycosylated proteins called mucins. Mucins regulate pathogen adhesion, growth, and virulence, and the glycans are vital for these functions. We analyzed intestinal mucin O-glycans on mucins from control and full-fat extruded soy-bean-fed (known to cause enteritis) Arctic charr using liquid chromatography-tandem mass spectrometry. In total, 56 glycans were identified on Arctic charr intestinal mucins, with a high prevalence of core-5-type and sialylated O-glycans. Disialic-acid-epitope-containing structures including NeuAcα2,8NeuAc, NeuAc(Gc)α2,8NeuGc(Ac), and NeuGcα2,8NeuGc were the hallmark of Arctic charr intestinal mucin glycosylation. Arctic charr fed with soy bean meal diet had lower (i) number of structures detected, (ii) interindividual variation, and (iii) N-glycolylneuraminic-acid-containing glycans compared with control Arctic charr. Furthermore, Aeromonas salmonicida grew less in response to mucins from inflamed Arctic charr than from the control group. The Arctic charr glycan repertoire differed from that of Atlantic salmon. In conclusion, the loss of N-glycolylneuraminic acid may be a biomarker for inflammation in Arctic char, and inflammation-induced glycosylation changes affect host-pathogen interactions.

Intranasal immunization with recombinant Trichinella spiralis serine protease elicits protective immunity in BALB/c mice.

The aim of this study was to observe the intestinal mucosal/systemic responses triggered by intranasal vaccination using recombinant Trichinella spiralis serine protease (rTsSP) and its capacity to elicit immune protection against larva challenge in a murine model. rTsSP coupled with cholera toxin B subunit (CTB) was used to vaccinate mice via intranasal route. The results revealed that intranasal vaccination with rTsSP plus CTB elicited significantly intestinal local sIgA response and a TsSP-specific systemic antibody response in vaccinated mice. Furthermore, more goblet cells/acidic mucins and IgA-secreting cells were observed in jejunum from vaccinated mice. Anti-rTsSP immune serum strongly recognized the cuticle of various worm stages (muscle larva, intestinal infective larva and adult worm). The level of IFN-γ, IL-4 and IL-10 of rTsSP-vaccinated mice was significantly elevated relative to CTB and PBS control groups. The vaccinated mice exhibited a 71.10% adult reduction at 9 days pi and a 62.10% muscle larva reduction at 42 days pi following larva challenge. Additionally, vaccination with rTsSP also dampened intestinal T. spiralis development and decreased the female fecundity. Our results showed that intranasal vaccination using rTsSP adjuvanted with CTB triggered significantly local sIgA response and systemic concurrent Th1/Th2 response that induced an obvious protection against Trichinella infection.

Profiling and Structural Characterization of High Neu5Gc or Sulfate-containing O-glycans from Hyla Rabbit Intestinal Mucin.

Intestinal mucins constitute the major component of the mucus covering the epithelium of the gastrointestinal tract, thereby forming a barrier against microbial colonization. Rabbits are bred in large numbers worldwide, with little known about intestinal O-glycosylation despite this insight being crucial to the understanding of host-pathogen interactions. In the present study, a major mucin-type glycopeptide (RIF6) of hyla rabbit intestine was isolated and the O-glycans were extensively characterized based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with bioinformatics approaches. Thirty-three O-glycans were identified, and most of them were sulfated or sialylated glycans. It was worth noting that Neu5Gc-containing structures within sialylated O-glycans accounted for 91%, which were extremely different from that of other species including humans, mice, chickens, etc. Sulfated glycans accounted for 58%, unique disufated and sulfated-sialylated glycans were also detected in rabbit intestinal mucin. These structural characterization reflected species diversity and may provide deeper insights into explaining the adaptability of hyla rabbit to the environment.

Reversible Condensation of Mucins into Nanoparticles.

Mucins are high molar mass glycoproteins that assume an extended conformation and can assemble into mucus hydrogels that protect our mucosal epithelium. In nature, the challenging task of generating a mucus layer, several hundreds of micrometers in thickness, from micrometer-sized cells is elegantly solved by the condensation of mucins inside vesicles and their on-demand release from the cells where they suddenly expand to form the extracellular mucus hydrogel. We aimed to recreate and control the process of compaction for mucins, the first step toward a better understanding of the process and creating biomimetic in vivo delivery strategies of macromolecules. We found that by adding glycerol to the aqueous solvent, we could induce drastic condensation of purified mucin molecules, reducing their size by an order of magnitude down to tens of nanometers in diameter. The condensation effect of glycerol was fully reversible and could be further enhanced and partially stabilized by cationic cross-linkers such as calcium and polylysine. The change of structure of mucins from extended molecules to nano-sized particles in the presence of glycerol translated into macroscopic rheological changes, as illustrated by a dampened shear-thinning effect with increasing glycerol concentration. This work provides new insight into mucin condensation, which could lead to new delivery strategies mimicking cell release of macromolecules condensed in vesicles such as mucins and heparin.

Genome-Wide Survey of Pseudomonas aeruginosa PA14 Reveals a Role for the Glyoxylate Pathway and Extracellular Proteases in the Utilization of Mucin.

Chronic airway infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. Although this bacterium has been extensively studied for its virulence determinants, biofilm growth, and immune evasion mechanisms, comparatively little is known about the nutrient sources that sustain its growth in vivo Respiratory mucins represent a potentially abundant bioavailable nutrient source, although we have recently shown that canonical pathogens inefficiently use these host glycoproteins as a growth substrate. However, given that P. aeruginosa, particularly in its biofilm mode of growth, is thought to grow slowly in vivo, the inefficient use of mucin glycoproteins may be relevant to its persistence within the CF airways. To this end, we used whole-genome fitness analysis, combining transposon mutagenesis with high-throughput sequencing, to identify genetic determinants required for P. aeruginosa growth using intact purified mucins as a sole carbon source. Our analysis reveals a biphasic growth phenotype, during which the glyoxylate pathway and amino acid biosynthetic machinery are required for mucin utilization. Secondary analyses confirmed the simultaneous liberation and consumption of acetate during mucin degradation and revealed a central role for the extracellular proteases LasB and AprA. Together, these studies describe a molecular basis for mucin-based nutrient acquisition by P. aeruginosa and reveal a host-pathogen dynamic that may contribute to its persistence within the CF airways.

A simplified chromatographic approach to purify commercially available bovine submaxillary mucins (BSM).

In this study, a simple purification protocol is developed to reduce the bovine serum albumin (BSA) content in commercially available bovine submaxillary mucin (BSM). This involved purification of the BSM by one-column anion-exchange chromatography protocol resulting in BSM with greatly reduced BSA content and homogeneously distributed size, and in a high yield of ∼43% from BSM as received from the manufacturer. The purity and composition of commercially acquired BSM were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry, which verified that BSA is the most abundant nonmucinous protein component. The purification effect was evident from a significantly altered circular dichroism (CD) spectrum of BSM after anion-exchange chromatography.

Aeromonas salmonicida binds differentially to mucins isolated from skin and intestinal regions of Atlantic salmon in an N-acetylneuraminic acid-dependent manner.

Aeromonas salmonicida subsp. salmonicida infection, also known as furunculosis disease, is associated with high morbidity and mortality in salmonid aquaculture. The first line of defense the pathogen encounters is the mucus layer, which is predominantly comprised of secreted mucins. Here we isolated and characterized mucins from the skin and intestinal tract of healthy Atlantic salmon and studied how A. salmonicida bound to them. The mucins from the skin, pyloric ceca, and proximal and distal intestine mainly consisted of mucins soluble in chaotropic agents. The mucin density and mucin glycan chain length from the skin were lower than were seen with mucin from the intestinal tract. A. salmonicida bound to the mucins isolated from the intestinal tract to a greater extent than to the skin mucins. The mucins from the intestinal regions had higher levels of sialylation than the skin mucins. Desialylating intestinal mucins decreased A. salmonicida binding, whereas desialylation of skin mucins resulted in complete loss of binding. In line with this, A. salmonicida also bound better to mammalian mucins with high levels of sialylation, and N-acetylneuraminic acid appeared to be the sialic acid whose presence was imperative for binding. Thus, sialylated structures are important for A. salmonicida binding, suggesting a pivotal role for sialylation in mucosal defense. The marked differences in sialylation as well as A. salmonicida binding between the skin and intestinal tract suggest interorgan differences in the host-pathogen interaction and in the mucin defense against A. salmonicida.

O-Linked glycome and proteome of high-molecular-mass proteins in human ovarian cancer ascites: Identification of sulfation, disialic acid and O-linked fucose.

The O-linked glycosylation of the main acidic high-molecular-weight glycoprotein from ascites fluid from patients with ovarian cancer were analyzed. The O-linked oligosaccharides were shown to consist of mainly highly sialylated core 1 and 2 structures with a smaller amount of sulfated core 2 structures. These structures were shown to be able to be further extended into small keratan sulfate (KS)-type oligosaccharides with up to four N-acetyllactosamine units. Proteomic studies of the acidic fraction of ascites fluid from patients with ovarian cancer showed that this fraction was enriched in proteoglycans. Among them, lumican, agrin, versican and dystroglycans were potential candidates, with threonine- and serine-rich domains that could carry a significant amount of O-linked glycosylation, including also the O-linked KS. Glycomic analysis using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) also showed that the disialic acid NeuAc-NeuAc- was frequently found as the terminating structure on the O-linked core 1 and 2 oligosaccharides from one ascites sample. Also, a small amount of the epidermal growth factor (EGF)-associated O-linked fucose structure Gal-GlcNAc-Fucitol was detected with and without sialic acid in the LC-MS/MS analysis. Candidate proteins containing O-linked fucose were suggested to be proteoglycan-type molecules containing the O-linked fucose EGF consensus domain.

The role of crude saliva and purified salivary mucins in the inhibition of the Human Immunodeficiency Virus type 1.

BACKGROUND: Sub-Saharan Africa is the world's worst HIV-AIDS affected region. More interventions to manage this pandemic are urgently required. Transmission of the virus through an exchange of saliva is rarely known to occur. This project sought to verify statistically previous findings in our laboratory, that crude saliva from uninfected individuals together with its purified mucin components inhibited HIV-1, whilst mucins from infected saliva did not show this inhibition, in an in vitro assay. METHODS: Saliva was extracted in 4 M guanidinium hydrochloride and proteolytic inhibitors at pH 6.5, followed by the isolation of MUC5B and MUC7 by Sepharose 4B gel filtration and further purification of these mucins by density-gradient ultra-centrifugation in caesium chloride. Agarose gel electrophoresis, Western blotting and amino acid compositional analysis determined the size, purity and identity of the mucins. The inhibitory activity of crude saliva and purified MUC5B and MUC7, from HIV negative (n=20) and HIV positive (n=20) donors, was tested by their incubation with subtype C HIV-1 and subsequent infection of peripheral blood mononuclear cells (PBMCs). PCR was done on tandem repeat regions of MUC5B and MUC7 DNA to investigate whether any association existed between gene polymorphism and susceptibility to infection. RESULTS: There was an inter-individual variation in the amounts of MUC5B and MUC7 in saliva. In contrast to previous studies, crude saliva and purified mucins from both HIV negative and HIV positive individuals inhibited the infection of HIV-1 in an in vitro assay. DNA analysis of the tandem repeat regions of MUC5B and MUC7 revealed no difference between groups. CONCLUSIONS: Crude saliva and its mucins, MUC5B and MUC7, from both uninfected controls and HIV positive individuals inhibited HIV-1 in an in vitro assay.

Interfacial interaction between transmembrane ocular mucins and adhesive polymers and dendrimers analyzed by surface plasmon resonance.

PURPOSE: Development of the first in vitro method based on biosensor chip technology designed for probing the interfacial interaction phenomena between transmembrane ocular mucins and adhesive polymers and dendrimers intended for ophthalmic administration. METHODS: The surface plasmon resonance (SPR) technique was used. A transmembrane ocular mucin surface was prepared on the chip surface and characterized by QCM-D (Quartz Crystal Microbalance with Dissipation) and XPS (X-ray photoelectron spectroscopy). The mucoadhesive molecules tested were: hyaluronic acid (HA), carboxymethyl cellulose (CMC), hydroxypropylmethyl cellulose (HPMC), chitosan (Ch) and polyamidoamine dendrimers (PAMAM). RESULTS: While Ch originated interfacial interaction with ocular transmembrane mucins, for HA, CMC and HPMC, chain interdiffusion seemed to be mandatory for bioadherence at the concentrations used in ophthalmic clinical practise. Interestingly, PAMAM dendrimers developed permanent interfacial interactions with transmembrane ocular mucins whatever their surface chemical groups, showing a relevant importance of co-operative effect of these multivalent systems. Polymers developed interfacial interactions with ocular membrane-associated mucins in the following order: Ch(1 %) > G4PAMAM-NH(2)(2 %) = G4PAMAM-OH(2 %) > G3.5PAMAM-COOH(2 %)>> CMC(0.5 %) = HA(0.2 %) = HPMC(0.3 %). CONCLUSIONS: The method proposed is useful to discern between the mucin-polymer chemical interactions at molecular scale. Results reinforce the usefulness of chitosan and dendrimers as polymers able to increase the retention time of drugs on the ocular surface and hence their bioavailability.

Identification of blood-protein carriers of the CA 19-9 antigen and characterization of prevalence in pancreatic diseases.

The current best serum marker for pancreatic cancer, CA 19-9, detects a carbohydrate antigen on multiple protein carriers. Better knowledge of the protein carriers of the CA 19-9 antigen in various disease states may lead to improved diagnostic tests. To identify proteins that carry the CA 19-9 antigen, we immunoprecipitated the CA 19-9 antigen from pooled sera and identified the associated proteins using MS. Among the high-confidence identifications, we confirmed the presence of the CA 19-9 antigen on Apolipoprotein B-100 by antibody arrays and Western blot and on kininogen, ARVCF, and Apolipoprotein E by antibody arrays. We characterized the frequency and levels of the CA 19-9 antigen on the four proteins across various patient groups (pancreatic cancer, pancreatitis, and healthy controls) using antibody arrays. Nearly, 10-25% of the subjects showed elevations of the antigen on each protein, but the elevations were not associated with disease state or total CA 19-9 levels. These results contribute to our knowledge of the carrier proteins of an important functional glycan and the rate at which the glycan is displayed. This work also demonstrates a strategy for using the complementary methods of MS and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.

Purified mucins in drug delivery research.

One of the main challenges in the field of drug delivery remains the development of strategies to efficiently transport pharmaceuticals across mucus barriers, which regulate the passage and retention of molecules and particles in all luminal spaces of the body. A thorough understanding of the molecular mechanisms, which govern such selective permeability, is key for achieving efficient translocation of drugs and drug carriers. For this purpose, model systems based on purified mucins can contribute valuable information. In this review, we summarize advances that were made in the field of drug delivery research with such mucin-based model systems: First, we give an overview of mucin purification procedures and discuss the suitability of model systems reconstituted from purified mucins to mimic native mucus. Then, we summarize techniques to study mucin binding. Finally, we highlight approaches that made use of mucins as building blocks for drug delivery platforms or employ mucins as active compounds.

The comparative inhibitory potency of salivary mucins against human immunodeficiency virus type 1.

MUC5B and MUC7 salivary mucins are reported to inhibit HIV-1 entry into target cells in vitro; however, their relative inhibitory potencies have not been quantitively compared. There is also conflicting evidence regarding whether HIV-1 infection diminishes mucins' inhibitory efficacy. We explored the effect of donor HIV-1 status upon the anti-HIV-1 potency of purified MUC5B and MUC7 while comparing their relative inhibitory potential using a pseudovirus-based neutralization assay. HIV status of sample donors had no detectable effect on HIV-1 inhibition by salivary mucins. MUC5B (median IC50 50 µg/ml, IQR 10-116 µg/ml) exhibited significantly more potent HIV-1 inhibition than MUC7 (median IC50 458 µg/ml, IQR 192->2000 µg/ml; Mann-Whitney U p < 0.0001). We suggest that larger size, gel-forming properties and extensive glycosylation of MUC5B allow more effective binding and aggregation of viral particles. MUC5B is also more abundant in the saliva and is therefore likely to make a substantially greater contribution to it's anti-HIV-1 properties.

Identification of oral squamous cell carcinoma markers MUC2 and SPRR1B downstream of TANGO.

PURPOSE: Transport and Golgi organization protein 1 (TANGO) promotes angiogenesis and lymphangiogenesis in oral squamous cell carcinoma (OSCC). To elucidate the underlying mechanisms, this study aims to identify and characterize elements downstream of TANGO that mediate its involvement in OSCC. METHODS: In this study, microarray analysis compared gene expression between control and TANGO-repressed HSC3 cells. Protein expression in 213 OSCC tissue samples was analyzed immunohistochemically. RESULTS: TANGO repression decreased or increased expression of Mucin 20 (MUC20) and small proline-rich protein 1B (SPRR1B), respectively. MUC20 increased the growth and invasiveness of OSCC cells via altered matrix metalloproteinase (MMP)-2 and E-cadherin expression and c-met phosphorylation. MUC20 induced angiogenesis and lymphangiogenesis by activating vascular endothelial growth factors A and C. In well-differentiated OSCC, SPRR1B expression was high (P = 0.0091) and correlated with keratinization markers and promoted proliferation by inducing mitogen-activated protein kinase p38 phosphorylation. MUC20 expression correlated significantly with clinical stage (P = 0.0024), lymph node metastasis (P = 0.0036), and number of blood and lymph vessels (P < 0.0001). MUC20-expressing cases had a significantly worse prognosis than non-expressing cases (P < 0.0001). CONCLUSION: MUC20 and SPRR1B located downstream of TANGO may be useful molecular markers for OSCC.

Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts.

BACKGROUND: We have previously shown that MUC5B and MUC7 mucins from saliva of HIV negative individuals inhibit HIV-1 activity by 100% in an in vitro assay. The purpose of this subsequent study was to investigate whether MUC5B and MUC7 from saliva of HIV patients or with full blown AIDS had a similar inhibitory activity against the virus. METHODS: Salivary MUC5B and MUC7 from HIV patients with different CD4 counts (< 200, 200-400 and > 400) were incubated with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). Cells were then cultured and viral replication was measured by a qualitative p24 antigen assay. The size, charge and immunoreactivity of mucins from HIV negative and positive individuals was also analysed by SDS-PAGE, Western blot and ELISA respectively. RESULTS: It was shown that irrespective of their CD4 counts both MUC5B and MUC7 from HIV patients, unlike the MUC5B and MUC7 from HIV negative individuals, did not inhibit HIV-1 activity. Size, charge and immunoreactivity differences between the mucins from HIV negative and positive individuals and among the mucins from HIV patients of different CD4 count was observed by SDS-PAGE, Western blot and ELISA. CONCLUSIONS: Purified salivary mucins from HIV positive patients do not inhibit the AIDS virus in an in vitro assay. Although the reason for the inability of mucins from infected individuals to inhibit the virus is not known, it is likely that there is an alteration of the glycosylation pattern, and therefore of charge of mucin, in HIV positive patients. The ability to inhibit the virus by aggregation by sugar chains is thus diminished.

Mucin is not a rare finding in rosacea.

Mucin deposit is a feature that is not commonly mentioned in textbooks when talking about rosacea. Nevertheless, it is one of the prominent findings of a severe variant of the phymatous type of rosacea known as the fibrous type. We retrospectively investigated 20 cases of early stages of rosacea and examined the mucin deposit in them, with histochemical stains (Alcian Blue and Periodic Acid-Schiff). We found granulomas in 20% of these cases. Alcian Blue positive deposits of mucin were found in all cases with granulomas. The mucin was located in the granulomas (four cases) as well as in the infundibulum (one case). No deposits of mucin were evidenced in the dermis out of the granulomas, apart from the normal mucin of papillary and adventicial dermis. Periodic Acid-Schiff did not show any deposits in any case. Serologic lupus markers were negative in all patients with mucin deposits. We conclude that: (a) mucin is a common finding in granulomas of rosacea; (b) this mucin is probably not related to any progression to the mucinous variant of rhinophyma; (c) since discoid erythematosus lupus is a clinical differential of rosacea, it is important to be aware of the fact that mucin is a common finding in the granulomas, in order not to misdiagnose both entities.

High-throughput and high-sensitivity nano-LC/MS and MS/MS for O-glycan profiling.

Sensitive and fast methods for the profiling of biologically important molecules are highly demanded. Mucins are densely O-glycosylated glycoproteins found at mucosal surfaces and are of great medical interest. Here we describe sensitive methods for the analysis of O-glycans from mucins using gel electrophoresis, and chromatography by nanoLC on graphite columns and structural analysis by electrospray mass spectrometry on a linear trap mass spectrometer.

NMR study on a novel mucin from jellyfish in natural abundance, Qniumucin from Aurelia aurita.

A novel mucin (qniumucin), which we recently discovered in jellyfish, was investigated by several NMR techniques. Almost all the peaks in the (13)C and proton NMR spectra were satisfactorily assigned to the amino acids in the main chain and to the bridging GalNAc, the major sugar in the saccharide branches. The amino acid sequence in the tandem repeat part (-VVETTAAP-) was reconfirmed by the cross-peaks between alpha protons and carbonyl carbons in the HMBC spectrum. A connectivity analysis around the O-glycoside bond (GalNAc-Thr) was also performed, and detailed information on the local configuration was obtained by the DPFGSE-NOE-HSD technique. The strategy and the results described in this paper can be extended to the structural analysis of general O-glycan chains, which are more complex than the present mucin. NMR analyses reveal the simple structure of qniumucin extracted by the present protocol, and the homogeneity and purity of qniumucin are probably the result of it being extracted from jellyfish, a primitive animal.