Paul R. Crocker

A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration

Following nerve injury, axons in the CNS do not normally regenerate. It has been shown that CNS myelin inhibits neurite outgrowth, though the nature of the molecules responsible for this effect are not known. Here, we demonstrate that the myelin-associated glycoprotein (MAG), a transmembrane protein of both CNS and PNS myelin, strongly inhibits neurite outgrowth from both developing cerebellar and adult dorsal root ganglion (DRG) neurons in vitro. This inhibition is reversed by an anti-MAG antibody. In contrast, MAG promotes neurite outgrowth from newborn DRG neurons. These results suggest that MAG may be responsible, in part, for the lack of CNS nerve regeneration in vivo and may influence, both temporally and spatially, regeneration in the PNS.

Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily

BACKGROUND Protein-carbohydrate interactions are believed to be important in many biological processes that involve cell-cell communication. Apart from the selectins, the only well-characterized vertebrate sialic acid-dependent adhesion molecules are CD22 and sialoadhesin; CD22 is a member of the immunoglobulin superfamily that is expressed by B lymphocytes and sialoadhesin is a macrophage receptor. The recent cloning of the gene encoding sialoadhesin has shown that it is also immunoglobulin-like. Both proteins share sequence similarity with the myelin-associated glycoprotein, an adhesion molecule of oligodendrocytes and Schwann cells that has been implicated in the process of myelination, raising the important question of whether myelin-associated glycoprotein is also a sialic acid-binding protein. RESULTS We have investigated the binding properties of these three receptors when expressed either in monkey COS cells or as chimaeric proteins containing the Fc portion of human immunoglobulin G. We demonstrate that, like sialoadhesin and CD22, myelin-associated glycoprotein mediates cell adhesion by binding to cell-surface glycans that contain sialic acid. We have dissected the specificities of these three adhesins further: whereas sialoadhesin binds equally to the sugar moieties NeuAc alpha 2-->3Gal beta 1-->3(4)GlcNAc or NeuAc alpha 2-->3Gal beta 1-->3GalNAc, myelin-associated glycoprotein recognizes only NeuAc alpha 2-->3Gal beta 1-->3GalNAc and CD22 binds specifically to NeuAc alpha 2-->6Gal beta 1-->4GlcNAc. Furthermore, we show that the recognition of sialylated glycans on the surfaces of particular cell types leads to the selective binding of sialoadhesin to neutrophils, myelin-associated glycoprotein to neurons and CD22 to lymphocytes. CONCLUSIONS Our findings demonstrate that a subgroup of the immunoglobulin superfamily can mediate diverse biological processes through recognition of specific sialylated glycans on cell surfaces. We propose that this subgroup of proteins be called the sialoadhesin family.

Carbohydrate recognition systems: functional triads in cell-cell interactions.

Considerable progress is being made in our understanding of the molecular basis for mammalian carbohydrate recognition systems. Selectins, related proteins and sialoadhesins are carbohydrate-binding proteins which serve as receptors in the orchestration of innate and acquired immune responses, inflammation and other forms of cell-cell communication. Protein structural studies and gain-of-function and loss-of-function mutations are providing clues to ways in which the receptors interact with monosaccharide elements of the oligosaccharide ligands. Binding experiments using oligosaccharides on lipid or protein carriers indicate that modes of presentation such as the clustered state and the manner of display on proteins are crucial factors determining whether a functional triad of receptor and ligand + carrier (counter-receptor) is formed.

Isolation of the gene for McLeod syndrome that encodes a novel membrane transport protein

McLeod syndrome is an X-linked multisystem disorder characterized by abnormalities in the neuromuscular and hematopoietic systems. We have assembled a cosmid contig of 360 kb that encompasses the McLeod gene locus. A 50 kb deletion was detected by screening DNA from patients with radiolabeled whole cosmids, and two transcription units were identified within this deletion. The mRNA expression pattern of one of them, designated as XK, correlates closely to the McLeod phenotype. XK encodes a novel protein with structural characteristics of prokaryotic and eukaryotic membrane transport proteins. Nucleotide sequence analysis of XK from two unrelated McLeod patients has identified point mutations at conserved splice donor and acceptor sites. These findings provide direct evidence that XK is responsible for McLeod syndrome.

Identification and Characterization of a Novel Siglec, Siglec-7, Expressed by Human Natural Killer Cells and Monocytes

We describe the characterization of sialic acid-binding Ig-like lectin-7 (siglec-7), a novel member of the siglec subgroup of the immunoglobulin superfamily. A full-length cDNA encoding siglec-7 was isolated from a human primary dendritic cell cDNA library. Siglec-7 is predicted to contain three extracellular immunoglobulin-like domains that comprise an N-terminal V-set domain and two C2-set domains, a transmembrane region and a cytoplasmic tail containing two tyrosine residues embodied in immunoreceptor tyrosine-based inhibition motif-like motifs. Overall, siglec-7 exhibited a high degree of sequence similarity to genes encoding CD33 (siglec-3), siglec-5, OBBP1/siglec-6, and OBBP-like protein and mapped to the same region on chromosome 19q13.3. When siglec-7 was expressed on COS or Chinese hamster ovary cells, it was able to mediate high levels of sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates, suggesting that it may be involved in cell-cell interactions. Among human peripheral blood leukocytes, siglec-7 was found to be present at low levels on granulocytes, intermediate levels on monocytes, and relatively high levels on a major subset of natural killer cells and a minor subset of CD8+ T cells. Immunoprecipitation experiments indicated that siglec-7 is expressed as a monomer of ∼65 kDa.

Modifications of cell surface sialic acids modulate cell adhesion mediated by sialoadhesin and CD22.

An increasing number of mammalian cell adhesion molecules, including sialoadhesion, CD22 and the family of selectins, have been found to bind cell surface glycoconjugates containing sialic acids. Here we describe how the structural diversity of this sugar influences cell adhesion mediated by the related molecules sialoadhesin and CD22 in murine macrophages and B-cells respectively. We show that the 9-O-acetyl group of Neu5,9Ac2 and theN-glycoloyl residue of Neu5Gc interfere with sialoadhesin binding. In contrast, CD22 binds more strongly to Neu5Gc compared to Neu5Ac. Of two synthetic sialic acids tested, only CD22 bound theN-formyl derivative, whereas aN-trifluoroacetyl residue was accepted by sialoadhesin. The potential significance for the regulation of sialic acid dependent cell adhesion phenomena is discussed.

The Sialoadhesins--a family of sialic acid-dependent cellular recognition molecules within the immunoglobulin superfamily.

For many years evidence has accumulated that sialic acids function in cellular interactions either by masking or as a recognition site. However, receptors or adhesion molecules mediating such functions between eukaryotic cells were unknown until about 5 years ago, when it was found that the members of the Selectin family mediate adhesion of leukocytes to specific endothelia through binding to sialylated glycans like sialyl Lewisx. More recently, the Sialoadhesin family of sialic acid-dependent adhesion molecules was defined within the superfamily of immunoglobulin-like molecules. So far, it has been shown that sialoadhesin (Sn), CD22, CD33, the myelin-associated glycoprotein (MAG) and the Schwann cell myelin protein (SMP) belong to this family. In contrast to the Selectins, these proteins are associated with diverse biological processes, i.e. hemopoiesis, neuronal development and immunity. In this review their properties, carbohydrate specificities and potential biological functions are discussed. Finally, we provide perspectives with respect to the nature of ligands, implications of sialic acid modifications and future research.

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.

Novel cell surface adhesion receptors involved in interactions between stromal macrophages and haematopoietic cells

Immunocytochemical staining of tissues with the mouse macrophage-specific monoclonal antibody, F4/80, has shown that large numbers of stromal macrophages are present in adult and foetal haematopoietic tissues. Macrophage plasma membrane processes are seen to establish extensive associations with myeloid and erythroid cells in adult bone marrow and with developing erythroblasts in foetal liver, suggestive of local trophic interactions. To explore the nature of these interactions, methods were developed for isolation of resident bone marrow macrophages (RBMM) and foetal liver macrophages (FLM). Following collagenase digestion of bone marrow or foetal liver, clusters were obtained which were composed of one or more central macrophages surrounded by proliferating haematopoietic cells. After attachment of clusters to glass coverslips, adherent macrophages could be stripped free of haematopoietic cells by pipetting in the absence of divalent cations. The purified RBMM, but not FLM, expressed a novel haemagglutinin, which mediated binding, without ingestion, of large numbers of unopsonized sheep erythrocytes by a divalent cation-independent mechanism. In view of the possibility that this sheep erythrocyte receptor (SER) could interact with a homologous ligand on mouse bone marrow cells, its properties were examined. SER was found to be a lectin-like protein which recognized protease-resistant sialylated glycoconjugates on sheep erythrocytes. The expression of SER was restricted to certain stromal tissue macrophages and was low or absent on monocytes and macrophages obtained from serous cavities. High levels of SER could be induced on elicited peritoneal macrophages by cultivation in mouse serum and the induced receptor was found to mediate low-avidity binding of murine bone marrow cells with characteristics indistinguishable from those seen for binding of sheep erythrocytes. However, maximal binding of bone marrow cells to RBMM depended on a distinct, divalent cation-dependent adhesion system. Using erythroblasts as a ligand, FLM were selected to explore the properties and expression of this adhesion receptor, the erythroblast receptor (EbR). Similar to SER, EbR did not mediate ingestion, and was restricted in its expression to foetal and adult stromal tissue macrophages. Unlike SER, EbR activity was not affected by neuraminidase treatment of the ligand and the receptor was not induced on peritoneal macrophages cultured in mouse serum. EbR appears to be a novel cell adhesion receptor because it was unaffected by inhibitors of several previously described cell adhesion molecules, including the fibronectin receptor. Future studies will attempt to explore the functional significance of these two receptors in macrophage-haematopoietic cell interactions.

The potential role of sialoadhesin as a macrophage recognition molecule in health and disease

Sialoadhesin is a macrophage-restricted transmembrane glycoprotein of 185 kDa that mediates cell–cell interactions through recognition of Neu5Acα2,3Gal in glycoconjugates. The extracellular region of sialoadhesin is composed of seventeen immunoglobulin-like domains, of which the amino-terminal two are highly-related structurally and functionally to the amino-terminal domains of CD22, myelin associated glycoprotein and CD33. These proteins, collectively known as the sialoadhesin family, are able to mediate sialic acid-dependent binding with distinct specificities for both the type of sialic acid and its linkage to subterminal sugars. In this review we discuss our recent studies on sialoadhesin and suggest how this molecule may contribute to a range of macrophage functions, both under normal conditions as well as during inflammatory reactions. Abbreviations: Ig, immunoglobulin; CEA, carcinoembryonic antigen; MAG, myelin associated glycoprotein; SMP Schwann cell myelin protein; mAb, monoclonal antibody; Chinese hamster ovary (CHO); UTR, untranslated region