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Background: A high proportion of patients with Crohn's disease (CD) over time develop complications like fistulae and strictures, requiring surgery. We tested a panel of antiglycan antibodies for predicting the occurrence of complications and CD-related surgery in an adult patient cohort. Methods: Serum samples of 149 CD patients of the German inflammatory bowel disease (IBD) network were tested for the presence of anti-laminarin IgA (Anti-L), anti-chitin IgA (Anti-C), anti-chitobioside IgA (ACCA), anti-laminaribioside IgG (ALCA), anti-mannobioside IgG (AMCA), and anti-Saccaromyces cerevisiae IgG (gASCA) carbohydrate antibodies by enzyme-linked immunosorbent assay (ELISA) (IBDX® panel, Glycominds, Lod, Israel) in a blinded fashion. Clinical data were available on occurrence of complicated disease or CD-related surgery as well as disease activity, onset, and location. Results: The median follow-up of the patients without any previous complication or surgery at time of sample procurement was 53.7 months. Overall, 26.3% developed a complication and 17.1% underwent CD-related surgery, respectively. Positivity for gASCA, AMCA, ACCA, and Anti-L alone or an increasing frequency of positive serum antibodies independently predicted a faster progression toward a more severe disease course. Once a complication or surgery had occurred only positivity for Anti-L or more than 3 markers out of the whole panel indicated progression to an additional surgery or complication. The antibody status of most patients remained stable over time. Conclusions: This is the first study showing the clinical value of serum antiglycan antibodies for prediction of a more complicated disease course in adult patients with CD. (Inflamm Bowel Dis 2010)

Background: We tested a panel of novel serological anti-glycan antibodies including the previously unpublished anti-laminarin IgA (Anti-L) and anti-chitin IgA (Anti-C) carbohydrate antibodies for the presence in Crohn's disease (CD) patients, diagnosis and differentiation of CD, association with complicated disease behavior, and marker stability over time. Methods: The presence of Anti-L, Anti-C, anti-chitobioside IgA (ACCA), anti-laminaribioside IgG (ALCA), anti-mannobioside IgG (AMCA), and anti-Saccaromyces cervisiae IgG (gASCA) carbohydrate antibodies were tested in serum samples from 824 participants (363 CD, 130 ulcerative colitis [UC], 74 other gastrointestinal diseases, and 257 noninflammatory bowel/gastrointestinal disease controls) of the German IBD-network by enzyme-linked immunosorbent assay (ELISA; Glycominds, Lod, Israel) and for perinuclear antineutrophil cytoplasmic antibody (pANCA) by immunofluorescence. Results: In all, 77.4% of the CD patients were positive for at least 1 of the anti-glycan antibodies. gASCA or the combination of gASCA/pANCA remained most accurate for the diagnosis of CD, but the combined use of the antibodies improved differentiation of CD from UC. Several single markers as well as an increasing antibody response were independently linked to a severe disease phenotype, as shown for the occurrence of complications, CD-related surgery, early disease onset, and ileal disease location. This was observed for both quantitative and qualitative antibody responses. The antibody status remained stable over time in most IBD patients. Conclusions: A panel of anti-glycan antibodies including the novel Anti-L and Anti-C may aid in differentiation of CD from UC, is associated with complicated CD behavior and IBD-related surgery, and is stable over time in a large patient cohort. Inflamm Bowel Dis 2009

The serum level of IgM antibodies against Glc(α1,4)Glc(α) (GAGA4) is higher in relapsing remitting multiple sclerosis (RRMS) compared to other neurological disease (OND) patients and healthy controls (HC). Detecting the level of anti-GAGA4 antibody by enzyme immunoassay and total IgM, we confirmed that anti-GAGA4 IgM can differentiate RRMS from OND patients and HC. Moreover, secondary progressive MS (SPMS) and RRMS patients have similar levels of anti-GAGA4 demonstrating the biomarker's presence throughout the disease. Interestingly, the anti-GAGA4 assay may also differentiate between primary progressive MS (PPMS) and RRMS/SPMS patients, since nearly all PPMS patients were negative for the assay.

A rapid and reproducible method was developed to detect and quantify carbohydrate-mediated cell adhesion to glycans arrayed on glass slides. Monosaccharides and oligosaccharides were covalently attached to glass slides in 1.7-mm-diameter spots (200 spots/slide) separated by a Teflon gasket. Primary chicken hepatocytes, which constitutively express a C-type lectin that binds to nonreducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-microL aliquots on the glycosylated spots. After incubating to allow cell adhesion, nonadherent cells were removed by immersing the slide in phosphate buffered saline, inverting, and centrifuging in a sealed custom acrylic chamber so that cells on the derivatized spots were subjected to a uniform and controlled centrifugal detachment force while avoiding an air-liquid interface. After centrifugation, adherent cells were fixed in place and detected by fluorescent imaging. Chicken hepatocytes bound to nonreducing terminal GlcNAc residues in different linkages and orientations but not to nonreducing terminal galactose or N-acetylgalactosamine residues. Addition of soluble GlcNAc (but not Gal) prior to incubation reduced cell adhesion to background levels. Extension of the method to CD4+ human T-cells on a 45-glycan diversity array revealed specific adhesion to the sialyl Lewis x structure. The described method is a robust approach to quantify selective cell adhesion using a wide variety of glycans and may contribute to the repertoire of tools for the study of glycomics.

Internalization of membrane proteins involves their recruitment into plasma membrane clathrin-coated pits, with which they are thought to interact by binding to AP-2 adaptor protein complexes. To investigate the interactions of membrane proteins with coated pits at the cell surface, we applied image correlation spectroscopy to measure directly and quantitatively the clustering of influenza hemagglutinin (HA) protein mutants carrying specific cytoplasmic internalization signals. The HA system enables direct comparison between isolated internalization signals, because HA itself is excluded from coated pits. The studies presented here provide, for the first time, a direct quantitative measure for the degree of clustering of membrane proteins in coated pits at the cell surface. The degree of clustering depended on the strength of the internalization signal and on the integrity of the clathrin lattices and correlated with the internalization rates of the mutants. The clustering of the HA mutants fully correlated with their ability to co-precipitate alpha-adaptin from whole cells, the first such demonstration for a membrane protein that is not a member of the epidermal growth factor receptor family. Furthermore, both the clustering in coated pits and the co-precipitation with alpha-adaptin were dramatically reduced in the cold, suggesting that low temperature can interfere with the sorting of proteins into coated pits. In addition to the specific results reported here, the general applicability of the image correlation spectroscopy approach to study any process involving the clustering or oligomerization of membrane receptors at the cell surface is discussed.

Measurements of the lateral mobility of native and mutated membrane proteins, combined with treatments that alter clathrin lattice structure, are capable of characterizing their interactions with coated pits in live cells (Fire, E., Zwart, D. E., Roth, M. G., and Henis, Y. I. (1991) J. Cell Biol. 115, 1585-1594). To explore the dependence of these interactions on the internalization signal and the aggregation state of the protein, we have extended this approach to investigate the interactions between coated pits and several influenza hemagglutinin (HA) mutants, which differ in the internalization signals in their short cytoplasmic tails. The lack of internalization signals in the trimeric wild-type HA enables a direct comparison between specific internalization signals introduced singly in each mutant. We have selected for these studies HA mutants that showed different internalization rates and varied in their tendency to aggregate into complexes larger than trimers. Our results indicate that the mode of interaction with coated pits (transient association-dissociation versus stable entrapment) depends on the internalization signal and affects the internalization efficiency. Mutants that contain a strong internalization signal and undergo fast endocytosis were entrapped in coated pits for the entire duration of the lateral mobility measurement, suggesting stable association with (slow dissociation from) coated pits. A mutant with a suboptimal internalization signal, which was internalized 10-fold slower, exhibited transient interactions with coated pits. Both types of interactions disappeared or were significantly reduced upon disruption of the clathrin lattices under hypertonic conditions, and were modulated following the "freezing" of coated pits by cytosol acidification. Unlike the dependence on the cytoplasmic internalization signal, the interactions with coated pits did not depend on the aggregation state (measured by sucrose gradient centrifugation after solubilization in n-octylglucoside) of the mutants.

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.

Protein ubiquitination is an important regulator of cytokine-activated signal transduction pathways and hematopoietic cell growth. Protein ubiquitination is controlled by the coordinate action of ubiquitin-conjugating enzymes and deubiquitinating enzymes. Recently a novel family of genes encoding growth-regulatory deubiquitinating enzymes (DUB-1 and DUB-2) has been identified.DUBs are immediate-early genes and are induced rapidly and transiently in response to cytokine stimuli. By means of polymerase chain reaction amplification with degenerate primers for theDUB-2 complementary DNA, 3 murine bacterial artificial chromosome (BAC) clones that contain DUB gene sequences were isolated. One BAC contained a novel DUB gene(DUB-2A) with extensive homology to DUB-2. LikeDUB-1 and DUB-2, the DUB-2A gene consists of 2 exons. The predicted DUB-2A protein is highly related to other DUBs throughout the primary amino acid sequence, with a hypervariable region at its C-terminus. In vitro, DUB-2Ahad functional deubiquitinating activity; mutation of its conserved amino acid residues abolished this activity. The 5′ flanking sequence of the DUB-2A gene has a hematopoietic-specific functional enhancer sequence. It is proposed that there are at least 3 members of the DUB subfamily (DUB-1, DUB-2,and DUB-2A) and that different hematopoietic cytokines induce specific DUB genes, thereby initiating a cytokine-specific growth response.

Cells maintain a complex traffic of membrane moving between secretory and endocytic organelles that have distinct protein compositions. This traffic requires mechanisms of sorting proteins into membrane domains that travel to specific destinations, and in the case of mobile proteins such as cell-surface receptors, multiple sorting events are required as the proteins circulate through various organelles. Considerable effort has been directed towards identifying the molecular interactions responsible for sorting membrane proteins (1–3), but our knowledge of the process is still limited.

The Novel Anti-Glycan Antibodies Anti-L and Anti-C in Conjunction with ALCA, ACCA, gASCA and AMCA Predict Early Development of Fistulae, Stenoses and Surgery in Patients with Crohn's Disease: A Prospective Analysis Florian Rieder, Alexandra Wolf, Stephan Schleder, Andrea Dirmeier, Ulrike Strauch, Florian Obermeier, Larissa Spector, Ella Fire, Jennifer Yarden, Gerhard Rogler, Nir Dotan, Frank Klebl