Biomedical subjects

Ingemar Carlstedt

Publications and source records attributed to Ingemar Carlstedt.

10 recordsLinked to original sources

PubMed2004

Effects of pH on Helicobacter pylori binding to human gastric mucins: identification of binding to non-MUC5AC mucins.

Helicobacter pylori causes gastritis, peptic ulcer disease and gastric cancer. The microbe is found in the gastric mucus layer where a pH gradient ranging from acidic in the lumen to neutral at the cell surface is maintained. The aim of the present study was to investigate the effects of pH on H. pylori binding to gastric mucins from healthy individuals. At pH 3, all strains bound to the most charged MUC5AC glycoform and to a putative mucin of higher charge and larger size than subunits of MUC5AC and MUC6, irrespective of host blood-group. In contrast, at pH 7.4 only Le(b)-binding BabA-positive strains bound to Le(b)-positive MUC5AC and to smaller mucin-like molecules, including MUC1. H. pylori binding to the latter component(s) seems to occur via the H-type-1 structure. All strains bound to a proteoglycan containing chondroitin sulphate/dermatan sulphate side chains at acidic pH, whereas binding to secreted MUC5AC and putative membrane-bound strains occurred both at neutral and acidic pH. The binding properties at acidic pH are thus common to all H. pylori strains, whereas mucin binding at neutral pH occurs via the bacterial BabA adhesin and the Le(b) antigen/related structures on the glycoprotein. Our work shows that microbe binding to membrane-bound mucins must be considered in H. pylori colonization, and the potential of these glycoproteins to participate in signalling events implies that microbe binding to such structures may initiate signal transduction over the epithelial layer. Competition between microbe binding to membrane-bound and secreted mucins is therefore an important aspect of host-microbe interaction.

Bacterial Adhesion↗

PubMed2004

Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin.

Adherence by Helicobacter pylori increases the risk of gastric disease. Here, we report that more than 95% of strains that bind fucosylated blood group antigen bind A, B, and O antigens (generalists), whereas 60% of adherent South American Amerindian strains bind blood group O antigens best (specialists). This specialization coincides with the unique predominance of blood group O in these Amerindians. Strains differed about 1500-fold in binding affinities, and diversifying selection was evident in babA sequences. We propose that cycles of selection for increased and decreased bacterial adherence contribute to babA diversity and that these cycles have led to gradual replacement of generalist binding by specialist binding in blood group O-dominant human populations.

ABO Blood-Group System↗

PubMed2004

Rhesus monkey gastric mucins: oligomeric structure, glycoforms and Helicobacter pylori binding.

Mucins isolated from the stomach of Rhesus monkey are oligomeric glycoproteins with a similar mass, density, glycoform profile and tissue localization as human MUC5AC and MUC6. Antibodies raised against the human mucins recognize those from monkey, which thus appear to be orthologous to those from human beings. Rhesus monkey muc5ac and muc6 are produced by the gastric-surface epithelium and glands respectively, and occur as three distinct glycoforms. The mucins are substituted with the histo blood-group antigens B, Le(a) (Lewis a), Le(b), Le(x), Le(y), H-type-2, the Tn-antigen, the T-antigen, the sialyl-Le(x) and sialyl-Le(a) structures, and the expression of these determinants varies between individuals. At neutral pH, Helicobacter pylori strains expressing BabA (blood-group antigen-binding adhesin) bind Rhesus monkey gastric mucins via the Le(b) or H-type-1 structures, apparently on muc5ac, as well as on a smaller putative mucin, and binding is inhibited by Le(b) or H-type-1 conjugates. A SabA (sialic acid-binding adhesin)-positive H. pylori mutant binds to sialyl-Le(x)-positive mucins to a smaller extent compared with the BabA-positive strains. At acidic pH, the microbe binds to mucins substituted by sialylated structures such as sialyl-Le(x) and sialylated type-2 core, and this binding is inhibited by DNA and dextran sulphate. Thus mucin- H. pylori binding occurs via at least three different mechanisms: (1) BabA-dependent binding to Le(b) and related structures, (2) SabA-dependent binding to sialyl-Le(x) and (3) binding through a charge-mediated mechanism to sialylated structures at low pH values.

Animals↗

PubMed2003

Sequential GC/MS analysis of sialic acids, monosaccharides, and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives.

A GC/MS procedure was developed for the analysis of all major constituents of glycoproteins. The rationale for this approach is that by using GC/MS analysis of the constituents as heptafluorobutyrate derivatives, it was possible to quantitatively determine the sialic acid, monosaccharide, fatty acids (when present), and the amino acid composition with the sample remaining in the same reaction vessel during the entire procedure. A mild acid hydrolysis was used to liberate sialic acids and was followed by formation of methyl-esters of heptafluorobutyrate (HFB) derivatives. After GC/MS analysis of sialic acids, the remaining material was submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. After GC/MS analysis of the monosaccharides, the sample was supplemented with norleucine (as internal standard) and hydrolyzed with 6 M HCl followed by the formation of isoamyl-esters of HFB derivatives and GC/MS analysis. His and Trp residues were modified during the step of acid-catalyzed methanolysis, but the resulting derivatives were stable during acid hydrolysis and quantitatively recovered by GC/MS analysis. As a result, all constituents of glycoproteins (sialic acids, monosaccharides (or di- and trisaccharides) and amino acids) are identified in the electron impact mode of ionization and quantified using three GC/MS analysis in the same chromatographic conditions and using a limited number of reagents, a considerable advantage over previous techniques. This method is very sensitive, all data (qualitative and quantitative) being obtained at the sub-nanomolar level of initial material.

Amino Acids↗

PubMed2003

Intracellular and interstitial expression of Helicobacter pylori virulence genes in gastric precancerous intestinal metaplasia and adenocarcinoma.

Gastric intestinal metaplasia (IM) and gastric cancer are associated with Helicobacter pylori, but the bacterium often is undetectable in these lesions. To unravel this apparent paradox, IM, H. pylori presence, and the expression of H. pylori virulence genes were quantified concurrently using histologic testing, in situ hybridization, and immunohistochemistry. H. pylori was detected inside metaplastic, dysplastic, and neoplastic epithelial cells, and cagA and babA2 expression was colocalized. Importantly, expression of cagA was significantly higher in patients with IM and adenocarcinoma than in control subjects. The preneoplastic "acidic" MUC2 mucin was detected only in the presence of H. pylori, and MUC2 expression was higher in patients with IM, dysplasia, and cancer. These novel findings are compatible with the hypothesis that all stages of gastric carcinogenesis are fostered by persistent intracellular expression of H. pylori virulence genes, especially cagA inside MUC2-producing precancerous gastric cells and pleomorphic cancer cells.

Adenocarcinoma↗

PubMed2003

Distribution of respiratory mucin proteins in human nasal mucosa.

OBJECTIVES/HYPOTHESIS: The upper respiratory tract is involved in many acute and chronic respiratory tract diseases that present with the symptom of mucus hypersecretion. Mucin genes that encode for the backbone of glycoproteins contribute to the viscoelastic property of airway mucus. We examined the cellular expression and distribution of two major respiratory mucus-forming glycoproteins, MUC5AC and MUC5B, in normal human nasal tissues. METHODS: Immunohistochemical analysis using polyclonal antibodies against the mucins MUC5AC and MUC5B was performed in normal human nasal tissues. RESULTS: An abundant staining of submucosal mucus gland and epithelial goblet cells for MUC5B was found. Immunohistochemical analysis of MUC5AC showed staining of surface epithelium goblet cells, whereas there was no staining of glandular cells. Comparison of the expression to lower airways revealed a similar pattern of expression of both mucins. CONCLUSIONS: The data in the present study demonstrated the localization of the two major respiratory mucin proteins in human nasal mucosa with a similar distribution of expression of MUC5AC and MUC5B in normal upper and lower airways. Mucin protein expression parallels that of mucin messenger RNA expression.

Acute Disease↗

PubMed2002

Gastric MUC5AC and MUC6 are large oligomeric mucins that differ in size, glycosylation and tissue distribution.

Gastric MUC5AC and MUC6 mucins were studied using polyclonal antibodies. Immunohistochemistry showed MUC5AC to originate from the surface epithelium, whereas MUC6 was produced by the glands. Mucins from the surface epithelium or glands of corpus and antrum were purified using CsCl/4M guanidinium chloride density-gradient centrifugation. MUC5AC appeared as two distinct populations at 1.4 and 1.3 g/ml, whereas MUC6, which was enriched in the gland tissue, appeared at 1.45 g/ml. Reactivity with antibodies against the Le(b) structure (where Le represents the Lewis antigen) followed the MUC5AC distribution, whereas antibodies against the Le(y) structure and reactivity with the GlcNAc-selective Solanum tuberosum lectin coincided with MUC6, suggesting that the two mucins are glycosylated differently. Rate-zonal centrifugation of whole mucins and reduced subunits showed that both gastric MUC5AC and MUC6 are oligomeric glycoproteins composed of disulphide-bond linked subunits and that oligomeric MUC5AC was apparently smaller than MUC6. A heterogeneous population of 'low-density' MUC5AC mucins, which were smaller than the 'high-density' ones both before and after reduction, reacted with an antibody against a variable number tandem repeat sequence within MUC5AC, suggesting that they represent precursor forms of this mucin. Following ion-exchange HPLC, both MUC5AC and MUC6 appeared as several distinct populations, probably corresponding to 'glycoforms' of the mucins, the most highly charged of which were found in the gland tissue.

Centrifugation, Density Gradient↗

PubMed2002

Strain- and blood group-dependent binding of Helicobacter pylori to human gastric MUC5AC glycoforms.

BACKGROUND & AIMS: In the stomach, Helicobacter pylori is found both in the mucus layer and adhering to the gastric epithelium. The aim of this study is to characterize the binding of H. pylori to human gastric mucins. METHODS: H. pylori strains that bind the Lewis(b) (Le(b)) structure (via the BabA adhesin) and/or sialylated structures, along with isogenic adhesion deletion mutants, were used to identify microbe-binding mucins. Gastric mucins from 5 healthy individuals, isolated by density-gradient centrifugation, were investigated for H. pylori binding at neutral pH using a microtiter-based technique. RESULTS: H. pylori strains that express the BabA adhesins were shown to bind to the MUC5AC mucin in individuals expressing the Le(b) antigen. Further fractionation with an ion-exchange chromatography revealed Le(b)-positive MUC5AC glycoforms that differed in their receptor properties for different H. pylori strains. None of the H. pylori strains studied bound to mucins from Le(b)-negative individuals. However, all strains bound to low-density, nonmucin, Le(b)-negative material on top of the gradients. CONCLUSIONS: Binding of H. pylori to human gastric MUC5AC isolated from healthy individuals is BabA dependent and mediated by the Le(b) structure presented by the mucin. However, the BabA adhesins demonstrate strain-dependent preference in binding to MUC5AC glycoforms substituted with Le(b), allowing for great interindividual variability in host-microbe interactions.

Adhesins, Bacterial↗

PubMed2002

Respiratory tract mucins: structure and expression patterns.

Goblet cells produce mainly MUC5AC, but also MUC5B and some MUC2 in apparently 'irritated' airways. MUC5B dominates in the submucosal glands although a little MUC5AC and MUC7 are usually present. MUC4 originates from the ciliated cells. After separation into a gel and a sol phase, lysozyme and lactoferrin are enriched in the salivary gel phase suggesting that mucus may act as a matrix for 'protective' proteins on the mucosal surface. A salivary MUC5B N-terminal fragment consistent with a cleavage event in the D' domain was detected with antibodies against various N-terminal peptide sequences suggesting that assembly of MUC5B occurs through a mechanism similar to that of the von Willebrand factor. Identification of additional cleavage sites C-terminal to the D' domain suggests that most of the N-terminal low-glycosylated part of MUC5B may be removed without affecting the oligomeric nature of the mucin. Possibly, the generation of mucins with different macromolecular properties through proteolytic 'processing' is one way of adapting the mucus polymer matrix to meet local physiological demands. Monomeric mucins that appear to turn over rapidly in the airway epithelium have been identified using radiolabelled mucin precursors. 'Shedding' of such mucins after microbe attachment may prevent colonization of epithelial surfaces.

Animals↗

PubMedDate not supplied

A high-density putative monomeric mucin is the major [35S]labelled macromolecular product of human colorectal mucins in organ culture.

We have studied the biosynthesis of mucins in organ cultures of human colon using isopycnic density-gradient centrifugation following pulse labelling with [(35)S]sulphate and [(3)H]-D-glucosamine. A high-density [(35)S]sulphate labelled component, of larger size than MUC2 monomers, appeared in the tissue and also in the medium. It was not degraded by reduction, trypsin digestion, digestion with chondroitin ABC lyase or heparan sulphate III lyase, but was cleaved into smaller fragments following alkaline borohydride treatment and appears to be a monomeric, mucin-like molecule containing a protease-resistant domain with a larger hydrodynamic volume than MUC2 monomers. Although this macromolecule incorporated much more radiolabel than MUC2, it was not detected using chemical analysis and thus appears to be a component with a high metabolic turnover present in a very small amount. Most of the [(3)H]-D-glucosamine label was associated with low-density material that was well separated from MUC2, which was poorly labelled. Most of MUC2 was associated with the tissue as an 'insoluble' complex. The amount of MUC2 remained constant and its associated radiolabel increased only slightly with time. Analysis of the MUC2 subunits from the reduced 'insoluble' complex showed the typical reduction-insensitive oligomers and confirmed that the radiolabel was associated with this mucin. The large size of the [(35)S]-labelled putative monomeric mucin makes it difficult to separate it from reduced insoluble complex MUC2. As a result, many studies of intestinal mucin synthesis and secretion in the past have most likely been performed on 'mixtures' of this mucin and MUC2 and are thus not possible to interpret as the metabolic behaviour of oligomeric mucins.

Centrifugation, Isopycnic↗