Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors.

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.

Minimal epitope for Mannitou IgM on paucimannose-carrying glycoproteins.

Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.

Chemoenzymatic Synthesis of N-glycan Positional Isomers and Evidence for Branch Selective Binding by Monoclonal Antibodies and Human C-type Lectin Receptors.

Here, we describe a strategy for the rapid preparation of pure positional isomers of complex N-glycans to complement an existing array comprising a larger number of N-glycans and smaller glycan structures. The expanded array was then employed to study context-dependent binding of structural glycan fragments by monoclonal antibodies and C-type lectins. A partial enzymatic elongation of semiprotected core structures was combined with the protecting-group-aided separation of positional isomers by preparative HPLC. This methodology, which avoids the laborious chemical differentiation of antennae, was employed for the preparation of eight biantennary N-glycans with Galß1,4GlcNAc (LN), GalNAcß1,4GlcNAc (LDN), and GalNAcß1,4[Fucα1,3]GlcNAc (LDNF) motifs presented on either one or both antennae. Screening of the binding specificities of three anti-LeX monoclonal IgM antibodies raised against S. mansoni glycans and three C-type lectin receptors of the innate immune system, namely DC-SIGN, DC-SIGNR, and LSECtin, revealed a surprising context-dependent fine specificity for the recognition of the glycan motifs. Moreover, we observed a striking selection of one individual positional isomer over the other by the C-type lectins tested, underscoring the biological relevance of the structural context of glycan elements in molecular recognition.

Fluoroacetamide Moieties as NMR Spectroscopy Probes for the Molecular Recognition of GlcNAc-Containing Sugars: Modulation of the CH-π Stacking Interactions by Different Fluorination Patterns.

We herein propose the use of fluoroacetamide and difluoroacetamide moieties as sensitive tags for the detection of sugar-protein interactions by simple 1 H and/or 19 F NMR spectroscopy methods. In this process, we have chosen the binding of N,N'-diacetyl chitobiose, a ubiquitous disaccharide fragment in glycoproteins, by wheat-germ agglutinin (WGA), a model lectin. By using saturation-transfer difference (STD)-NMR spectroscopy, we experimentally demonstrate that, under solution conditions, the molecule that contained the CHF2 CONH- moiety is the stronger aromatic binder, followed by the analogue with the CH2 FCONH- group and the natural molecule (with the CH3 CONH- fragment). In contrast, the molecule with the CF3 CONH- isoster displayed the weakest intermolecular interaction (one order of magnitude weaker). Because sugar-aromatic CH-π interactions are at the origin of these observations, these results further contribute to the characterization and exploration of these forces and offer an opportunity to use them to unravel complex recognition processes.

Chemo-Enzymatic Synthesis of (13)C Labeled Complex N-Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry.

Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of (13)C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was (13)C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.

Granulomatous slack skin-like clinical findings in Sézary syndrome.

Granulomatous slack skin (GSS) is a very rare condition that has been described as a variant of mycosis fungoides. It is characterized by the development of bulky and pendulous skin folds in flexural areas that are histologically formed by atypical T lymphocytes, histiocytes and giant cells. We report the case of a 37-year-old African-American female with history of Sézary syndrome (SS) that while on treatment for the disease and in a space of 1 month developed exorbitant slack folds in the axillae and cervical area mimicking GSS. The absence of giant cells and epithelioid granulomas in the biopsy ruled out this diagnosis. We report this peculiar SS presentation that clinically resembles GSS, but with histopathology that does not show the typical features of this condition. We also review the literature in regard to SS, GSS and granulomatous mycosis fungoides (GMF), particularly the existing criteria to differentiate these various entities.

Monitoring Glycan-Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH-π Interactions.

Detection of molecular recognition processes requires robust, specific, and easily implementable sensing methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan-protein interactions that involve N-acetylated sugars. Although difluoroacetamide has been used previously as a substituent in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc-containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal (1) H and (19) F atoms that allow both (1) H and (19) F NMR methods for easy and robust detection of molecular recognition processes involving GlcNAc- (or GalNAc-) moieties over a range of binding affinities. The CHF2 CONH- moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic-sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF3 CONH- analogue differs more significantly.

Synthesis and microarray-assisted binding studies of core xylose and fucose containing N-glycans.

The synthesis of a collection of 33 xylosylated and core-fucosylated N-glycans found only in nonmammalian organisms such as plants and parasitic helminths has been achieved by employing a highly convergent chemo-enzymatic approach. The influence of these core modifications on the interaction with plant lectins, with the human lectin DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin), and with serum antibodies from schistosome-infected individuals was studied. Core xylosylation markedly reduced or completely abolished binding to several mannose-binding plant lectins and to DC-SIGN, a C-type lectin receptor present on antigen presenting cells. Employing the synthetic collection of core-fucosylated and core-xylosylated N-glycans in the context of a larger glycan array including structures lacking these core modifications, we were able to dissect core xylose and core fucose specific antiglycan antibody responses in S. mansoni infection sera, and we observed clear and immunologically relevant differences between children and adult groups infected with this parasite. The work presented here suggests that, quite similar to bisecting N-acetylglucosamine, core xylose distorts the conformation of the unsubstituted glycan, with important implications for the immunogenicity and protein binding properties of complex N-glycans.

Toward the solid-phase synthesis of heparan sulfate oligosaccharides: evaluation of iduronic acid and idose building blocks.

Glycan arrays have been established as the premier technical platform for assessing the specificity of carbohydrate binding proteins, an important step in functional glycomics research. Access to large libraries of well-characterized oligosaccharides remains a major bottleneck of glycan array research, and this is particularly true for glycosaminoglycans (GAGs), a class of linear sulfated polysaccharides which are present on most animal cells. Solid-supported synthesis is a potentially powerful tool for the accelerated synthesis of relevant GAG libraries with variations in glycan sequence and sulfation pattern. We have evaluated a series of iduronic acid and idose donors, including a couple of novel n-pentenyl orthoester donors in the sequential assembly of heparan sulfate precursors from monosaccharide building blocks in solution and on a polystyrene resin. The systematic study of donor and acceptor performance up to the trisaccharide stage in solution and on the solid support have resulted in a general strategy for the solid-phase assembly of this important class of glycans.

Infantile acne treated with oral isotretinoin.

In contrast to adolescent acne, infantile acne (IA) is a rare condition with only a limited body of available literature. In this descriptive, retrospective study, we reviewed six cases from 2002 to 2010 treated with oral isotretinoin. The average age of onset was 6.16 months (range 0-21 mos). Consistent with the previous, limited literature, we found predominantly boys are affected, a predilection for the cheeks, and a polymorphic inflammatory morphology. Two patients had a family history of acne. All cases were successfully and safely treated with oral isotretinoin. The suggested treatment of childhood acne is similar to that of adolescents (graded according to the severity of the skin disease and risk of scarring). Oral isotretinoin appears to be an effective and safe treatment for severe IA.

Phacomatosis melanovascularis: a new example of non-allelic twin spotting.

We report an infant who had a combination of two rather common nevoid skin conditions, namely "telangiectatic nevus" or "capillary malformation", and "hyperpigmented mosaicism" or "segmental pigmentary disorder" in close apposition, occupying large areas of the body in a mosaic distribution. This association may be considered a further example of didymosis, for which we propose the term, "phacomatosis melanovascularis" to denote a possible non-allelic twin spotting condition. To our knowledge, this is the first case reported in the literature.

Erythema multiforme major associated with CMV infection in an immunocompetent patient.

BACKGROUND: Cytomegalovirus (CMV) infection usually remains asymptomatic in immunocompetent adults, and few cases of complicated disease in nonimmunosuppressed patients have been reported. Erythema multiforme (EM), an acute and self-limiting skin eruption characterized by a typical targetoid lesion that may also affect the mucosa, is a hypersensitivity reaction that usually occurs after herpes simplex virus infection or use of certain drugs and resolves without complications in healthy individuals. To our knowledge, CMV infection has been associated with EM in only six patients. OBJECTIVE: We present a case of an EM caused by CMV infection in a 35-year-old nonimmunosuppressed patient who was successfully treated with ganciclovir. CONCLUSION: Our report, like other similar reports found in the literature, suggests that CMV can trigger EM in apparently healthy individuals. Intravenous ganciclovir appears to be a good treatment option in these cases.

Multiple eruptive keratoacanthomas arising in a tattoo.

Keratoacanthomas are rapidly growing, keratinizing, epithelial neoplasms that tend to spontaneously involute and are rarely multiple or eruptive. There is still disagreement on whether or not this condition is a malignancy or a benign epidermal neoplasm; nevertheless, its appearance on tattoos has been reported in rare instances. When waiting for spontaneous involution is not an option, surgery is the preferred treatment. Other therapeutic modalities used for the treatment of this condition include radiotherapy; cryotherapy; laser therapy; and multiple intralesional, topical, and systemic agents. The authors report a patient who developed multiple, eruptive keratoacanthomas in the red ink portions of a tattoo and was successfully treated with acitretin.