Elastin-like hydrogel stimulates angiogenesis in a severe model of critical limb ischemia (CLI): An insight into the glyco-host response.

Critical limb ischemia (CLI) is characterized by the impairment of microcirculation, necrosis and inflammation of the muscular tissue. Although the role of glycans in mediating inflammation has been reported, changes in the glycosylation following muscle ischemia remains poorly understood. Here, a murine CLI model was used to show the increase of high mannose, α-(2, 6)-sialic acid and the decrease of hybrid and bisected N-glycans as glycosylation associated with the ischemic environment. Using this model, the efficacy of an elastin-like recombinamers (ELR) hydrogel was assessed. The hydrogel modulates key angiogenic signaling pathways, resulting in capillary formation, and ECM remodeling. Arterioles formation, reduction of fibrosis and anti-inflammatory macrophage polarization wa also induced by the hydrogel administration. Modulation of glycosylation was observed, suggesting, in particular, a role for mannosylation and sialylation in the mediation of tissue repair. Our study elucidates the angiogenic potential of the ELR hydrogel for CLI applications and identifies glycosylation alterations as potential new therapeutic targets.

Cartilage oligomeric matrix protein forms protein complexes with synovial lubricin via non-covalent and covalent interactions.

OBJECTIVE: Understanding the cartilage surface structure, lost in arthritic disease, is essential for developing strategies to effectively restore it. Given that adherence of the lubricating protein, lubricin, to the cartilage surface is critical for boundary lubrication, an interaction with cartilage oligomeric matrix protein (COMP) was investigated. COMP, an abundant cartilage protein, is known to be important for matrix formation. DESIGN: Synovial fluid (SF) from arthritic patients was used to detect possible COMP-lubricin complexes by immunological methods. Recombinant (RC) COMP and lubricin fragments were expressed to characterize this bonding and mass spectrometry employed to specifically identify the cysteines involved in inter-protein disulfide bonds. RESULTS: COMP-lubricin complexes were identified in the SF of arthritic patients by Western blot, co-immunoprecipitation and sandwich ELISA. RC fragment solid-phase binding assays showed that the C-terminal (amino acids (AA) 518-757) of COMP bound non-covalently to the N-terminal of lubricin (AA 105-202). Mass spectrometry determined that although cysteines throughout COMP were involved in binding with lubricin, the cysteines in lubricin were primarily focused to an N-terminal region (AA 64-86). The close proximity of the non-covalent and disulfide binding domains on lubricin suggest a two-step mechanism to strongly bind lubricin to COMP. CONCLUSION: These data demonstrate that lubricin forms a complex network with COMP involving both non-covalent and covalent bonds. This complex between lubricin and the cartilage protein COMP can be identified in the SF of patients with arthritis conditions including osteoarthritis (OA) and rheumatoid arthritis (RA).

Characterisation of lubricin in synovial fluid from horses with osteoarthritis.

REASON FOR PERFORMING STUDY: The glycoprotein lubricin contributes to the boundary lubrication of the articular cartilage surface. The early events of osteoarthritis involve the superficial layer where lubricin is synthesised. OBJECTIVES: To characterise the glycosylation profile of lubricin in synovial fluid from horses with osteoarthritis and study secretion and degradation of lubricin in an in vitro inflammation cartilage model. STUDY DESIGN: In vitro study. METHODS: Synovial fluid samples collected from horses with joints with normal articular cartilage and structural osteoarthritic lesions; with and without osteochondral fragments, were analysed for the lubricin glycosylation profiles. Articular cartilage explants were stimulated with or without interleukin-1ß for 25 days. Media samples collected at 3-day intervals were analysed by quantitative proteomics, western blot and enzyme-linked immunosorbent assay. RESULTS: O-glycosylation profiles in synovial fluid revealed both Core 1 and 2 O-glycans, with Core 1 O-glycans predominating. Synovial fluid from normal joints (49.5 ± 1.9%) contained significantly lower amounts of monosialylated Core 1 O-glycans compared with joints with osteoarthritis (53.8 ± 7.8%, P = 0.03) or joints with osteochondral fragments (57.3 ± 8.8%, P = 0.001). Additionally, synovial fluid from normal joints (26.7 ± 6.7%) showed higher amounts of disialylated Core 1 O-glycan than from joints with osteochondral fragments (21.2 ± 4.9%, P = 0.03). A C-terminal proteolytic cleavage site in lubricin was found in synovial fluid from normal and osteochondral fragment joints and in media from interleukin-1ß stimulated and unstimulated articular cartilage explants. CONCLUSIONS: This is the first demonstration of a change in the glycosylation profile of lubricin in synovial fluid from diseased equine joints compared with that from normal joints. We demonstrate an identical proteolytic cleavage site of lubricin both in vitro and in vivo. The reduced sialation of lubricin in synovial fluid from diseased joints may affect the boundary lubricating ability of the superficial layer of articular cartilage and could be one of the early events in the progression of osteoarthritis.

Mucins in the mucosal barrier to infection.

The mucosal tissues of the gastrointestinal, respiratory, reproductive, and urinary tracts, and the surface of the eye present an enormous surface area to the exterior environment. All of these tissues are covered with resident microbial flora, which vary considerably in composition and complexity. Mucosal tissues represent the site of infection or route of access for the majority of viruses, bacteria, yeast, protozoa, and multicellular parasites that cause human disease. Mucin glycoproteins are secreted in large quantities by mucosal epithelia, and cell surface mucins are a prominent feature of the apical glycocalyx of all mucosal epithelia. In this review, we highlight the central role played by mucins in accommodating the resident commensal flora and limiting infectious disease, interplay between underlying innate and adaptive immunity and mucins, and the strategies used by successful mucosal pathogens to subvert or avoid the mucin barrier, with a particular focus on bacteria.

Proteomic analysis of the genetic premature aging disease Hutchinson Gilford progeria syndrome reveals differential protein expression and glycosylation.

Proteomics has revealed differential protein expression and glycosylation in membrane proteins from premature aging Hutchinson-Gilford progeria syndrome fibroblasts (progeria). Progeria is a rare autosomal dominant genetic disorder of premature aging characterized by marked growth retardation and specific, progressive, premature senescent changes of the skin and other tissues. Affected children live to an average age of 13 years. The 1q20-24 region of chromosome 1 which codes for one of these proteins, lamin A/C, has previously been implicated by Brown et al. (1990) who described identical twins with progeria, where cytogenetic analysis showed an inverted insertion in the long arm of the chromosome in 70% of cells. Luengo et al. (2002) similarly reported an interstitial deletion of chromosome 1q23, in a 9-year-old patient with a classic clinical picture of progeria.

Fluticasone propionate aqueous nasal spray in pregnancy rhinitis.

Pregnancy rhinitis is a common condition with longstanding nasal congestion; troublesome for the mother, possibly also affecting the fetus. There is need for a safe, effective treatment. Nasal corticosteroids, indisputable in other types of rhinitis, have not been evaluated in pregnancy rhinitis. In this placebo-controlled, randomized, double-blind study with parallel groups, we evaluated the effect of 8 weeks of treatment with fluticasone propionate aqueous nasal spray in 53 women with pregnancy rhinitis. Daily symptom scores and nasal peak expiratory flow, as well as acoustic rhinometry before and after treatment, did not show any difference between the groups. Placebo resulted in 6/27 responders, compared with 5/26 for active treatment. There was no detectable influence on maternal cortisol as measured by morning S-cortisol and overnight 12-h-U-cortisol, or any difference in ultrasound measures of fetal growth or pregnancy outcome. Altogether, our study indicates no significant effects of the treatment described here.

Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.

Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, beta1,2 N-acetylglucosaminyltransferase I, and beta1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography-mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.

Identification of transient glycosylation alterations of sialylated mucin oligosaccharides during infection by the rat intestinal parasite Nippostrongylus brasiliensis.

The sialylation of the oligosaccharides from small-intestinal mucins during a 13-day infectious cycle was studied in Sprague-Dawley rats with the parasite Nippostrongylus brasiliensis. Sialic acid analysis and release, permethylation and analysis by GC-MS of the sialylated oligosaccharides isolated from the 'insoluble' mucin complex revealed a relative decrease (4-7-fold) of N-glycolylneuraminic acid compared with N-acetylneuraminic acid just before parasite expulsion. Northern blots showed that this effect was due to the decreased expression of a hydroxylase converting CMP-N-acetylneuraminic acid into CMP-N-glycolylneuraminic acid. Analysis of other rat strains showed that this parasite infection also caused the same effect in these animals. Detailed analysis of infected Sprague-Dawley rats revealed four sialylated oligosaccharides not found in the uninfected animals. These new oligosaccharides were characterized in detail and all shown to contain the trisaccharide epitope NeuAc/NeuGcalpha2-3(GalNAcbeta1-4)Galbeta1 (where NeuGc is N-glycolyl neuraminic acid). This epitope is similar to the Sd(a)- and Cad-type blood-group antigens and suggests that the infection causes the induction of a GalNAcbeta1-4 glycosyltransferase. This model for an intestinal infection suggests that the glycosylation of intestinal mucins is a dynamic process being modulated by the expression of specific enzymes during an infection process.

Nasal mucociliary transport in pregnancy.

Using the saccharin method, we performed tests of mucociliary function four times during pregnancy and once one month after delivery in 27 women. As the transport distance for saccharin varied from 37 to 65 mm, we used the transport speed for evaluation. Pregnancy rhinitis affects at least 20% of pregnancies. The mucociliary transport speed was higher in the group of women with pregnancy rhinitis, and was reduced during pregnancy in the group of women without that condition. We found no significant correlation between mucociliary transport speed and objectively registered nasal peak expiratory flow index. The pathophysiology of pregnancy rhinitis is not known, but is possibly multifactorial. The changes occurring in the nasal mucociliary transport system during normal pregnancy and in pregnancy rhinitis need further studies.

Liquid chromatography-electrospray mass spectrometry as a tool for the analysis of sulfated oligosaccharides from mucin glycoproteins.

An approach for analyzing sulfated oligosaccharide alditol mixtures by liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) is described. Two columns, an amino-bonded column and a porous graphitized carbon column (PGC) were used. Oligosaccharides were eluted with linear gradients of acetonitrile and water, with 5 mM ammonium hydrogencarbonate or formate buffers at a basic pH. The methods were evaluated on a mixture of sulfated oligosaccharide alditols prepared from mucin glycoproteins from pig stomach. Results from LC-ESI-MS of the mixture were compared with the structural information obtained by high energy collision fragmentation using fast atom bombardment tandem mass spectrometry (FAB-MS-MS). The separation ability of the two columns was also tested using a more complex mixture of sulfated oligosaccharides from pig colon, where several isomers were detected. The potential use of in-source collision-induced dissociation (CID) to gain sequence information of sulfated oligosaccharides was also evaluated. The major fragment ions obtained by in-source CID of the trisaccharide Hex-3HexNAcol6-HexNAc6-SO3 were sufficient for assigning the oligosaccharide sequence and the position of the sulfate group within the monosaccharide moiety. The LC-ESI-MS approach should be a valuable tool for characterization of mucin glycosylation and alterations during pathological conditions.

Detection of CD43 (leukosialin) in colon adenoma and adenocarcinoma by novel monoclonal antibodies against its intracellular domain.

CD43 is a leukocyte-associated sialoglycoprotein which is also expressed in human colon adenoma and carcinoma. To obtain monoclonal antibodies (MAbs) that would react with CD43 in a glycosylation-independent way, antibodies were raised against a peptide corresponding to a portion of the CD43 cytoplasmic domain. Hybridomas were screened on paraffin sections from CD43-positive colon tumours. The reactivity of the antibodies with CD43 was verified by Western blot analysis of lysate of CHO cells transfected with human CD43 cDNA and by immunoprecipitation of lysates from CD43+ cell lines. Epitope mapping of antibodies was done using overlapping heptameric peptides. A detailed characterisation of one of the novel antibodies (CD43-3A1) is presented. This antibody reacts with the CD43 protein regardless of its glycosylation in Western blot analysis, immunoprecipitation and immuno-histochemistry of paraffin sections. Immuno-histochemical analysis of paraffin sections from colon adenoma and carcinoma tissues as well as colon cancer cell lines revealed that CD43 was predominantly localised intracellularly, in contrast to leukocyte-type cells. The MAb reacted more efficiently with paraffin-embedded colon adenoma and carcinoma cells than previously characterised CD43-specific antibodies. This should facilitate the evaluation of a potential role of CD43 during cancer development.

Different O-glycosylation of respiratory mucin glycopeptides from a patient with cystic fibrosis.

The O-linked oligosaccharides from three fractions of highly glycosylated mucin glycopeptides obtained from sputum of a patient with cystic fibrosis were characterized and compared regarding size, composition, sequence and when possible linkage positions. Neutral and sialic acid-containing glycans were permethylated and analyzed by high-temperature GC-MS and MALDI-MS, showing more than 60 different oligosaccharides with a size of up to 15 monosaccharide units. Some of the observed oligosaccharides are novel for respiratory secretions, one being a trifucosylated heptasaccharide with the proposed structure: Fuc-Gal-4(Fuc-3)GlcNAc-(Fuc-)Gal-3GalNAcol. The glycosylation of two of the glycopeptide fractions was similar with regard to the neutral and sialylated oligosaccharides despite their different origins from the sol or gel phase. Analysis of the sulfated oligosaccharides by FAB-MS/MS indicated that the gel fraction contained C-6 linked sulfate groups while the two sol fractions also contained C-3 linked sulfate. The results suggest the presence of different glycosylated mucin domains, probably originating from different mucin glycoforms and/or apoproteins in the airway of cystic fibrosis patients.

The glycosylation of rat intestinal Muc2 mucin varies between rat strains and the small and large intestine. A study of O-linked oligosaccharides by a mass spectrometric approach.

The large glycosylated domains obtained from the rat intestinal mucin Muc2 were isolated from the large and small intestine of the inbred rat strains GOT-W and GOT-BW. The expression of the rat Muc2 in the large intestine was confirmed immunochemically and by Northern blotting. Released oligosaccharides were structurally characterized by gas chromatography-mass spectrometry (neutral and sialylated species) or by tandem mass spectrometry (sulfated species), and a total of 63 structures was assigned. The large intestinal oligosaccharides were found to be identical between the strains, while the small intestinal glycosylation differed. Until now, detailed structural analysis of oligosaccharides isolated from a single mucin core or mucin domain with different origin have not been performed, and the information of different mucin glycoforms has been limited to immunochemistry. Blood group A-determinants (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-, and structures related to the blood group Sda/Cad-related epitope NeuAc/NeuGcalpha1-3(GalNAcbeta1-4)Galbeta1-, were found in GOT-BW small intestine, and also in both large intestines. Blood group H-determinants and NeuAc/NeuGcalpha1-3Galbeta1- were found in all samples. Core 1 (Galbeta1-3GalNAcalpha1-), core 2 (Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-), core 3 (GlcNAcbeta1-3GalNAcalpha1-), and core 4 (GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1- were also found in all the samples. The large intestine were enriched in sulfated oligosaccharides and the small intestine contained higher amounts of sialylated species. Sulfation were found exclusively on C-6 of GlcNAc.

Mucus glycoproteins from pig gastric mucosa: identification ofdifferent mucin populations from the surface epithelium.

Pig gastric mucins were isolated from the surface epithelium of the cardiac region, corpus and antrum using density-gradient centrifugation after extraction in 6 M guanidinium chloride. In CsCl/0.5 M guanidinium chloride, mucins solubilized from the cardiac region appeared as a broad unimodal band at 1.52 g/ml whereas those from the corpus and antrum occurred as high- and low-density populations at 1.50 and 1.45 g/ml respectively. High-iron diamine reacted more strongly with the cardiac mucins and the high-density populations from corpus and antrum than with the two low-density ones. In keeping with this, approx. 60% of the oligosaccharides from the former mucins and 20% from the latter contained sulphate. All surface epithelial cells of the cardiac region stained with high-iron diamine, whereas in the corpus only the epithelium in the bottom of the pits reacted, suggesting that the high-density population from this region originates from these cells. Mucins from all regions were composed of subunits, each containing highly glycosylated domains. The mucins from the cardiac region were larger than those from the corpus and antrum, and reduced subunits as well as high-molecular-mass glycopeptides from the cardiac mucins were larger than the corresponding fragments from the other regions. Ion-exchange HPLC showed that reduced subunits from the cardiac mucins and the high-density populations from the corpus and antrum were more 'acidic' than reduced subunits from the two low-density ones. All mucins contained a 'neutral'fraction, in particular those from the antrum. Pig gastric mucus thus contains a number of distinctly different mucin populations varying in buoyant density, size, 'acidity', glycosylation, sulphation and tissue origin.

Glycosylation differences between pig gastric mucin populations: a comparative study of the neutral oligosaccharides using mass spectrometry.

Five mucin populations were isolated from the cardiac region,corpus and antrum of pig gastric mucosa. The released neutral oligosaccharides were permethylated and analysed using high-temperature gas chromatography-mass spectrometry (GC-MS) as well as matrix-assisted laser-desorption mass spectrometry (MALDI-MS). Thirty different oligosaccharides with up to six monosaccharide residues were characterized using both techniques, but the presence of an additional 49 structures was suggested on the basis of their molecular mass by MALDI-MS. Oligosaccharides based on core-1 (Galbeta1-3GalNAcalpha1-) and core-2 [Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-] structures were widely distributed, whereas core-3 structures (GlcNAcbeta1-3GalNAcalpha1-) were present only in mucins from the cardiac region and corpus, and core-4 structures [GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-] were present exclusively in mucins from the cardiac region. Furthermore the oligosaccharides from one of the mucins from the corpus were significantly longer than those from the other populations. The results illustrate vast structural diversity, but the relative abundances show only a few dominating structures, suggesting that many oligosaccharides may be quite rare in pig gastric mucins. Well-defined mucin populations with distinctly different glycosylation can thus be identified in pig stomach, suggesting that glycosylation of the large secreted mucins from this tissue is not a random event.

Molecular characterization of the large heavily glycosylated domain glycopeptide from the rat small intestinal Muc2 mucin.

The largest high-glycosylated domain, glycopeptide A, of the "insoluble' mucin complex of the rat small intestine has earlier been purified and characterized (Carlstedt et al., 1993, J Biol Chem 268: 18771-81). A rabbit antiserum raised against deglycosylated glycopeptide A was used to clone part of a mucin showing homology to the human MUC2 mucin (Hansson et al., 1994, Biochem Biophys Res Commun 198. 181-90). This serum specifically stained goblet cells (paranuclear) in the mouse small intestine. The size of the coding sequence of glycopeptide A was estimated by using reversed transcriptase PCR of mRNA from an inbred rat strain (GOT-W) using primers in the unique central and C-terminal parts of the proposed rat Muc2 sequences. The PCR and Southern blot of the PCR products showed a fragment of about 5.5 kb corresponding to about 1700 amino acids when the known Cys-rich sequences used for the primers were subtracted. This is slightly larger than the size estimated earlier by biochemical studies. The mRNA encoding the rat Muc2 was slightly smaller than the mRNA encoding the human MUC2 in a colorectal cell line. Although the size of glycopeptide A estimated from biochemical results and by PCR is not identical, the results obtained here further support that the "insoluble' mucin of the rat small intestine is encoded by the Muc2 gene. Most of the oligosaccharides in glycopeptide A were either neutral (40%) or sialylated (40%). The remaining ones were sulfated with the sulfate group attached to C-6 of N-acetylglucosamine linked to C-6 of the N-acetylgalactosaminitol as revealed by tandem mass spectrometry of the perdeuteroacetylated oligosaccharides. Eighteen oligosaccharides were found of which fourteen were characterized and found to be mostly novel. Our findings thus expand the current knowledge of the core peptide of the rat intestinal goblet cell mucin and provide a relatively complete picture of the glycosylation of a defined mucin domain.

Sulphated mucin oligosaccharides from porcine small intestine analysed by four-sector tandem mass spectrometry.

The fraction of sulphated oligosaccharide alditols isolated from mucin glycopeptides of porcine small intestine 'insoluble' mucin complex was analysed by negative-ion fast atom bombardment (FAB) tandem mass spectrometry. Collision-induced dissociation (CID) tandem mass spectra of native and peracetylated species were compared with standards of sulphated monosaccharides. The tandem mass spectra revealed structural information of the carbohydrate sequence and sulphate position. Negative-ion FAB ionization of the peracetylated sulphated oligo-saccharide alditols was at least three times more sensitive than that of the native sulphated oligosaccharide alditols, as revealed by comparing the signal-to-noise ratios, and allowed the detection of eleven compared with six pseudo-molecular ions. Fourteen structures were determined from the CID tandem mass spectra obtained. The main sulphation site was C-6 of an N-acetylglucosamine 6-linked to the N-acetylgalactosaminitol. C-3 of the N-acetylgalactosaminitol could be unsubstituted or extended with a series of up to three monosaccharide residues including blood group H determinants and blood group A determinants. Also, the sulphated N-acetylglucosamine could be further extended. The most abundant structure was a monosulphated trisaccharide with the sequence Gal-->3(SO3-->6GlcNAc-->6)GalNAcol. The sulphation at C-6 of N-acetylglucosamine seems to be a common feature for O-linked oligosaccharides, and has been described both for skeletal keratan sulphates and respiratory mucin oligosaccharides. Low-abundance ions were also detected from oligosaccharides with sulphation at C-3 of an amino sugar residue. This seems to be a novel sulphation site for mucin oligosaccharides.

Strategy for the investigation of O-linked oligosaccharides from mucins based on the separation into neutral, sialic acid- and sulfate-containing species.

A method for the separation of O-linked oligosaccharides into neutral, sialic acid-containing and sulfated species was applied to oligosaccharides released by alkaline borohydride from mucin glycopeptides from porcine small intestine. The released mixture of reduced oligosaccharides was applied to an anion exchange column, and the neutral oligosaccharides were collected as the unretarded fraction. A mixture of dimethyl sulfoxide and iodomethane was passed through the column to convert the sialic acid-containing oligosaccharides into methyl esters that were eluted and converted to methyl amides by methyl amine. Finally the sulfated oligosaccharide fraction was eluted with salt. The neutral and the derivatized sialic acid-containing oligosaccharides were analysed by gas chromatography-mass spectrometry after permethylation and the sulfated oligosaccharide fraction was analysed by high performance anion exchange chromatography.

Analysis of monosaccharide composition of mucin oligosaccharide alditols by high-performance anion-exchange chromatography.

A simple one-step method for the analysis of monosaccharides including galactosaminitol after acidic hydrolysis is described. The hydrolyzate was re-N-acetylated and analyzed by high-performance anion-exchange chromatography and the sugars were detected by a pulsed amperometric detector. The method was applied on a mixture of neutral oligosaccharides released from mucin glycopeptides of rat small intestine by alkaline borohydride. Sugars were detected down to the nanomole range and the results were compared with monosaccharide compositional analysis performed by gas chromatography of acetylated alditols.