Sørge Kelm

The amino-terminal immunoglobulin-like domain of sialoadhesin contains the sialic acid binding site. Comparison with CD22.

Sialoadhesin and CD22 are members of a recently characterized family of sialic acid-dependent adhesion molecules belonging to the immunoglobulin superfamily. Sialoadhesin is a macrophage-restricted receptor containing 17 extracellular Ig-like domains which recognizes oligosaccharides terminating in NeuAcα2-3Gal in N- and O-linked glycans. CD22 is a B cell-restricted receptor with seven Ig-like domains which selectively recognizes oligosaccharides terminating in NeuAcα2-6Gal in N-glycans. Sequence similarity between these proteins is highest within their first four amino-terminal Ig-like domains. Here we identify the domain(s) containing the binding sites of both molecules by generating a series of extracellular domain deletion mutants fused to the Fc portion of human IgG1. Binding activity was analyzed by solid phase cell adhesion assays and also by surface plasmon resonance using purified glycophorin and CD45 as ligands for sialoadhesin and CD22, respectively. For sialoadhesin, the amino-terminal V-set Ig-like domain was both necessary and sufficient to mediate sialic acid-dependent adhesion of the correct specificity. In contrast, for murine CD22, only constructs containing both the V-set domain and the adjacent C2-set domain were able to mediate sialic acid-dependent binding. These results are consistent with the sialic acid binding site for both proteins residing in the membrane distal V-set domain, but for CD22 a direct contribution in binding from the neighboring C2-set domain cannot be excluded.

Structure-Guided Design of Sialic Acid-Based Siglec Inhibitors and Crystallographic Analysis in Complex with Sialoadhesin

The Siglec family of receptors mediates cell surface interactions through recognition of sialylated glycoconjugates. The crystal structure of the N-terminal immunoglobulin-like domain of the Siglec sialoadhesin (SnD1) in complex with 2,3-sialyllactose has informed the design of sialic acid analogs (sialosides) that bind Siglecs with significantly enhanced affinities and specificities. Binding assays against sialoadhesin (Sn; Siglec-1), CD22 (Siglec-2), and MAG (Siglec-4) show a 10- to 300-fold reduction in IC(50) values (relative to methyl-alpha-Neu5Ac) for three sialosides bearing aromatic group modifications of the glycerol side chain: Me-alpha-9-N-benzoyl-amino-9-deoxy-Neu5Ac (BENZ), Me-alpha-9-N-(naphthyl-2-carbonyl)-amino-9-deoxy-Neu5Ac (NAP), and Me-alpha-9-N-(biphenyl-4-carbonyl)-amino-9-deoxy-Neu5Ac (BIP). Crystal structures of these sialosides in complex with SnD1 suggest explanations for the differences in specificity and affinity, providing further ideas for compound design of physiological and potentially therapeutic relevance.

Fibronectin is a binding partner for the myelin‐associated glycoprotein (siglec‐4a)

The myelin‐associated glycoprotein (MAG) mediates cell–cell interactions between myelinating glial cells and neurons. Here we describe the extracellular matrix glycoprotein fibronectin as a binding partner of MAG. It has been identified by affinity precipitation with MAG‐Fc from NG108‐15 cells and by microsequencing of two peptides derived from a 210‐kDa protein band. Western blot analysis showed that fibronectin is also present in MAG binding partners isolated from N2A (murine neuroblastoma) cells, rat brain and rat spinal cord. Different fibronectin isoforms have been isolated from brains of young and adult rats, indicating that the expression of MAG binding fibronectin changes during development.

From a library of MAG antagonists to nanomolar CD22 ligands.

Siglec‐2, also known as CD22, is involved in the regulation and survival of B‐cells and has been successfully targeted in cell depletion therapies with antibody‐based approaches. Sialic acid derivatives, already known to bind with high affinity to myelin‐associated glycoprotein (MAG, Siglec‐4), were screened for their binding affinity for CD22 by surface plasmon resonance. The best compound identified was further modified with various hydrophobic substituents at the 2‐, 5‐, and 9‐positions of the sialic acid scaffold, leading to nanomolar derivatives, of which ligand 17 b shows the most promising pharmacodynamic and pharmacokinetic profiles. Isothermal titration calorimetry measurements demonstrate that the binding is enthalpy driven. Interestingly, the thermodynamic fingerprints reveal an excellent correlation between gains in enthalpy and compensation by increased entropy costs. Moreover, 17 b exhibits a residence time in the range of a few seconds, clearly prolonged relative to residence times typically observed for carbohydrate–lectin interactions. Finally, initial tests regarding drug‐like properties of 17 b demonstrate the required high plasma protein binding yet a lack of oral availability, although its distribution coefficient (log D) is in the required range.

Examination of the Biological Role of the α(2→6)-Linked Sialic Acid in Gangliosides Binding to the Myelin-Associated Glycoprotein (MAG)

The tetrasaccharide 1, a substructure of ganglioside GQ1b alpha, shows a remarkable affinity for the myelin-associated glycoprotein (MAG) and was therefore selected as starting point for a lead optimization program. In our search for structurally simplified and pharmacokinetically improved mimics of 1, modifications of the core disaccharide, the alpha(2-->3)- and the alpha(2-->6)-linked sialic acid were synthesized. Biphenylmethyl and (S)-lactate were identified as suitable replacements for the alpha(2-->6)-linked sialic acid. Combined with a core modification and the earlier found aryl amide substituent in the 9-position of the alpha(2-->3)-linked sialic acid, high affinity MAG antagonists were identified. All mimics were tested in a competitive target-based binding assay, providing relative inhibitory potencies (rIP). Compared to the reference tetrasaccharide 1, the rIPs of the most potent antagonists 59 and 60 are enhanced nearly 400-fold. Their K(D)s determined in surface plasmon resonance experiments are in the low micromolar range. These results are in semiquantitative agreement with molecular modeling studies. This new class of glycomimetics will allow to validate the role of MAG in the axon regeneration process.

Expression of Hepatoma-derived growth factor family members in the adult central nervous system

BackgroundHepatoma-derived growth factor (HDGF) belongs to a polypeptide family containing five additional members called HDGF related proteins 1–4 (HRP-1 to -4) and Lens epithelial derived growth factor. Whereas some family members such as HDGF and HRP-2 are expressed in a wide range of tissues, the expression of others is very restricted. HRP-1 and -4 are only expressed in testis, HRP-3 only in the nervous system. Here we investigated the expression of HDGF, HRP-2 and HRP-3 in the central nervous system of adult mice on the cellular level by immunohistochemistry. In addition we performed Western blot analysis of various brain regions as well as neuronal and glial cell cultures.ResultsHDGF was rather evenly expressed throughout all brain regions tested with the lowest expression in the substantia nigra. HRP-2 was strongly expressed in the thalamus, prefrontal and parietal cortex, neurohypophysis, and the cerebellum, HRP-3 in the bulbus olfactorius, piriform cortex and amygdala complex. HDGF and HRP-2 were found to be expressed by neurons, astrocytes and oligodendrocytes. In contrast, strong expression of HRP-3 in the adult nervous system is restricted to neurons, except for very weak expression in oligodendrocytes in the brain stem. Although the majority of neurons are HRP-3 positive, some like cerebellar granule cells are negative.ConclusionThe coexpression of HDGF and HRP-2 in glia and neurons as well as the coexpression of all three proteins in many neurons suggests different functions of members of the HDGF protein family in cells of the central nervous system that might include proliferation as well as cell survival. In addition the restricted expression of HRP-3 point to a special function of this family member for neuronal cells.

A Novel Type of Macrothrombocytopenia Associated with a Defect in α2,3-Sialylation

We describe a novel type of human thrombocytopenia characterized by the appearance of giant platelets and variable neutropenia. Searching for the molecular defect, we found that neutrophils had strongly reduced sialyl-Lewis X and increased Lewis X surface expression, pointing to a deficiency in sialylation. We show that the glycosylation defect is restricted to α2,3-sialylation and can be detected in platelets, neutrophils, and monocytes. Platelets exhibited a distorted structure of the open canalicular system, indicating defective platelet generation. Importantly, patient platelets, but not normal platelets, bound to the asialoglycoprotein receptor (ASGP-R), a liver cell-surface protein that removes desialylated thrombocytes from the circulation in mice. Taken together, this is the first type of human thrombocytopenia in which a specific defect of α2,3-sialylation and an induction of platelet binding to the liver ASGP-R could be detected.

SUMOylation of the hepatoma‐derived growth factor negatively influences its binding to chromatin

Hepatoma‐derived growth factor is a nuclear targeted mitogen containing a PWWP domain that mediates binding to DNA. To date, almost nothing is known about the molecular mechanisms of the functions of hepatoma‐derived growth factor, its routes of secretion and internalization or post‐translational modifications. In the present study, we show for the first time that hepatoma‐derived growth factor is modified by the covalent attachment of small ubiquitin‐related modifier 1 (SUMO‐1), a post‐translational modification with regulatory functions for an increasing number of proteins. Using a basal SUMOylation system in Escherichia coli followed by a MALDI‐TOF‐MS based peptide analysis, we identified the lysine residue SUMOylated located in the N‐terminal part of the protein adjacent to the PWWP domain. Surprisingly, this lysine residue is not part of the consensus motif described for SUMOylation. With a series of hepatoma‐derived growth factor mutants, we then confirmed that this unusual location is also used in mammalian cells and that SUMOylation of hepatoma‐derived growth factor takes place in the nucleus. Finally, we demonstrate that SUMOylated hepatoma‐derived growth factor is not binding to chromatin, in contrast to its unSUMOylated form. These observations potentially provide new perspectives for a better understanding of the functions of hepatoma‐derived growth factor.

Biochemical characterization of trans-sialidase TS1 variants from Trypanosoma congolense

BackgroundAnimal African trypanosomiasis, sleeping sickness in humans and Nagana in cattle, is a resurgent disease in Africa caused by Trypanosoma parasites. Trans-sialidases expressed by trypanosomes play an important role in the infection cycle of insects and mammals. Whereas trans-sialidases of other trypanosomes like the American T. cruzi are well investigated, relatively little research has been done on these enzymes of T. congolense.ResultsBased on a partial sequence and an open reading frame in the WTSI database, DNA sequences encoding for eleven T. congolense trans-sialidase 1 variants with 96.3% overall amino acid identity were amplified. Trans-sialidase 1 variants were expressed as recombinant proteins, isolated and assayed for trans-sialylation activity. The purified proteins produced α2,3-sialyllactose from lactose by desialylating fetuin, clearly demonstrating their trans-sialidase activity. Using an HPLC-based assay, substrate specificities and kinetic parameters of two variants were characterized in detail indicating differences in substrate specificities for lactose, fetuin and synthetic substrates. Both enzymes were able to sialylate asialofetuin to an extent, which was sufficient to reconstitute binding sites for Siglec-4. A mass spectrometric analysis of the sialylation pattern of glycopeptides from fetuin revealed clear but generally similar changes in the sialylation pattern of the N-glycans on fetuin catalyzed by the trans-sialidases investigated.ConclusionsThe identification and characterization of a trans-sialidase gene family of the African parasite T. congolense has opened new perspectives for investigating the biological role of these enzymes in Nagana and sleeping sickness. Based on this study it will be interesting to address the expression pattern of these genes and their activities in the different stages of the parasite in its infection cycle. Furthermore, these trans-sialidases have the biotechnological potential to be used for enzymatic modification of sialylated glycoconjugates.

Binding and Inhibition Assays for Siglecs

Protein-carbohydrate interactions are believed to be important in many biological processes involving cellular communication. Although many of the experimental approaches for studying protein-carbohydrate interactions are similar to those commonly employed in protein-protein interactions, several important aspects have to be taken into account in binding assays with lectin-like receptors such as Siglecs, sialic acid-recognizing immunoglobulin-like lectins. This chapter describes experimental approaches using solid-phase assays with sialylated targets and complexed Siglec Fc-chimeras for binding studies or competitive inhibition assays. Such assays are useful to investigate various aspects of Siglec interactions with their carbohydrate-binding partners, such as the specificity and the molecular basis of sialic acid recognition.