Synthesis of a panel of carbon-13-labelled (glyco)sphingolipids

Medical Biochemistr

Bioinformatics and molecular modeling in glycobiology

The field of glycobiology is concerned with the study of the structure, properties, and biological functions of the family of biomolecules called carbohydrates. Bioinformatics for glycobiology is a particularly challenging field, because carbohydrates exhibit a high structural diversity and their chains are often branched. Significant improvements in experimental analytical methods over recent years have led to a tremendous increase in the amount of carbohydrate structure data generated. Consequently, the availability of databases and tools to store, retrieve and analyze these data in an efficient way is of fundamental importance to progress in glycobiology. In this review, the various graphical representations and sequence formats of carbohydrates are introduced, and an overview of newly developed databases, the latest developments in sequence alignment and data mining, and tools to support experimental glycan analysis are presented. Finally, the field of structural glycoinformatics and molecular modeling of carbohydrates, glycoproteins, and protein–carbohydrate interaction are reviewed

Structural and Functional Insights into Endoglin Ligand Recognition and Binding

Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-β receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-β signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted

Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity

The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/ or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and Cterminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn136 in V1 (T138N mutation) inconjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N139INN sequence, which ablates the overlapping Asn141-Asn142-Ser-Ser potential N-linked glycosylation sequons inV1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu593, Trp596 and Lys601. The 136 and/or 142glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection

Chemical and structural analysis of an antibody folding intermediate trapped during glycan biosynthesis

Human IgG Fc glycosylation modulates immunological effector functions such as antibody-dependent cellular cytotoxicity and phagocytosis. Engineering of Fc glycans therefore enables fine-tuning of the therapeutic properties of monoclonal antibodies. The N-linked glycans of Fc are typically complex-type, forming a network of noncovalent interactions along the protein surface of the Cγ2 domain. Here, we manipulate the mammalian glycan-processing pathway to trap IgG1 Fc at sequential stages of maturation, from oligomannose- to hybrid- to complex-type glycans, and show that the Fc is structurally stabilized following the transition of glycans from their hybrid- to complex-type state. X-ray crystallographic analysis of this hybrid-type intermediate reveals that N-linked glycans undergo conformational changes upon maturation, including a flip within the trimannosyl core. Our crystal structure of this intermediate reveals a molecular basis for antibody biogenesis and provides a template for the structure-guided engineering of the protein-glycan interface of therapeutic antibodies

Studies towards the total synthesis of solanoeclepin A: enantioselective synthesis of the right-hand substructure

Aardappelmoeheid wordt veroorzaakt door het aardappelcystenaaltje. Om deze parasiet op een natuurlijke manier te bestrijden is de natuurlijke wekstof (Solanoëclepine A) geïsoleerd. De parasieten komen uit zodra de aardappelplanten kleine hoeveelheden van deze stof uitscheiden in de lente. Ginger Lutteke wilde een route ontwikkelen om deze wekstof in het laboratorium te maken met behulp van chemische synthese. Het onderzoek leidde tot de ontwikkeling van een syntheseroute naar een gedeelte van de wekstof. Volgens Lutteke is verder onderzoek nodig om te kijken of dit gedeelte van de wekstof activiteit vertoont. De testen kunnen inzicht verschaffen in de relatie tussen de structuur van de wekstof en de activiteit. Dit zou uiteindelijk kunnen leiden tot de ontwikkeling van een selectief bestrijdingsmiddel voor het aardappelcystenaaltje

Enantioselective Approach to the Right-Hand Substructure of Solanoeclepin A

Item does not contain fulltextAn enantioselective synthesis of the right-hand substructure of solanoeclepin A has been developed. The key step was an intramolecular [2+2] photocycloaddition between an allene and a butenolide providing a methylenecyclobutane with three quaternary carbon atoms in a complex tetracyclic framework. Other crucial steps included an enantioselective Noyori transfer hydrogenation of a ketone, a diastereoselective silver-mediated silyl dienolate allylation, and a diastereoselective cyclopropanation of an allylic alcohol. The installation of the bridgehead methyl group by reduction of the lactone moiety proved to be troublesome

Formal Synthesis of Solanoeclepin A: Enantioselective Allene Diboration and Intramolecular 2+2 Photocycloaddition for the Construction of the Tricyclic Core

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