Kurt Drickamer

Trimeric structure of a C-type mannose-binding protein.

BACKGROUNDnMannose-binding proteins (MBPs) are C-type (Ca(2+)-dependent) animal lectins found in serum. They recognize cell-surface oligosaccharide structures characteristic of pathogenic bacteria and fungi, and trigger the neutralization of these organisms. Like most lectins, MBPs display weak intrinsic affinity for monovalent sugar ligands, but bind avidly to multivalent ligands.nnnRESULTSnWe report physical studies in solution and the crystal structure determined at 1.8 A Bragg spacings of a trimeric fragment of MBP-A, containing the carbohydrate-recognition domain (CRD) and the neck domain that links the carboxy-terminal CRD to the collagen-like portion of the intact molecule. The neck consists of a parallel triple-stranded coiled coil of alpha-helices linked by four residues to the CRD. The isolated neck peptide does not form stable helices in aqueous solution. The previously characterized carbohydrate-binding sites lie at the distal end of the trimer and are separated from each other by 53 A.nnnCONCLUSIONSnThe carbohydrate-binding sites in MBP-A are too far apart for a single trimer to bind multivalently to a typical mammalian high-mannose oligosaccharide. Thus MBPs can recognize pathogens selectively by binding avidly only to the widely spaced, repetitive sugar arrays on pathogenic cell surfaces. Sequence alignments reveal that other C-type lectins are likely to have a similar oligomeric structure, but differences in their detailed organization will have an important role in determining their interactions with oligosaccharides.

Ca2+-dependent carbohydrate-recognition domains in animal proteins

Many Ca 2+ -dependent (C-type) animal lectins contain a common sequence motif, with 14 invariant and 18 highly conserved residues distributed over discrete carbohydrate-recognition domains of 115–130 amino acids. Domains that display the C-type carbohydrate-recognition domain motif are found in an increasing number of proteins, although only some are known to bind carbohydrate. Progress is being made towards making deductions about the structure and activity of these domains from their sequences.

Evolution of Ca2+-dependent Animal Lectins

Publisher Summary This chapter discusses the evolution of Ca 2+ -dependent animal lectins. Animal lectins have been identified in various contexts, both as a result of direct searches for proteins with selective sugar-binding activities, and less systematically in the course of investigation of biological recognition processes. In spite of the enormous diversity of animal lectins, analysis of their primary structures reveals that most fall into relatively few categories. In all cases, sugar-binding activity is associated with discrete protein modules, of 115-140 amino acids, termed carbohydrate-recognition domains (CRDs). Three major groups of animal lectins (P, S, and C-types) contain CRDs with distinct sequence motifs. Animal lectins, grouped together are based on structural considerations also share certain important functional properties. The mannose-6-phosphate receptors (P-type lectins) and thiol-dependent p-galactoside-binding lectins (Stype lectins) each have relatively restricted specificity for a particular type of sugar structure. The C-type lectins, in contrast, are characterized not by a common type of sugar ligand, but by a shared dependence on Ca2 + for sugar-binding activity.

Clearing up glycoprotein hormones.

Among the multitude of carbohydrate structures found in nature, the ones that seem most likely to bear specific information are the complex oligosaccharides attached to extracellular proteins and to proteins and lipids at the surface of eukaryotic cells. Although our understanding of the roleof the saccharide portionsof such glycoconjugates is still incomplete, results presented in this issue of Cell provide important insight into their functions. To appreciate the significance of these findings, it is useful to reflect on the unique properties of complex oligosaccharides that may make them especially suited for conveying information in particular situations. Multiple Roles ot Giycosyiation The idea that cell surface sugars form an important part of the identity of cells has been given a substantial boost by the recent demonstration that oligosaccharide recognition by the sefectins is an important first step in adhesion of leukocytes to selected portions of the endothelium (Stoolman, 1989). The cell surface oligosaccharides present a high concentration of target ligands, which facilitates the initial interaction between stationary and rapidly moving cells. High affinity of individual saccharide-receptor interactions is not so important as high valency. The initial sugar-mediated binding need only be sufficiently tight to allow time for more specific protein-protein adhesion systems to consolidate the interaction. Although the generality of this type of function for cell surface sugars remains to be demonstrated, we at least have a provocative paradigm. In contrast to the plasma membrane, each serum glycoprotein carries one or at most a few oligosaccharides. The sugar units on these glycoproteins must, therefore, function in a more individualistic manner. One possibility is that the conjugation of saccharides to proteins allows proteins to achieve unique conformations that facilitate their intrinsic activities, such as catalysis, ligand binding, or receptor binding. Indeed, the ability of specific saccharide structures to modify the intrinsic activity of individual serum proteins has now been well established (Parekh, 1991). However, given the subtle variety of function that can be achieved with polypeptidesalone, it is hard to imagine how sugars could add sufficiently to the repertoire to be worth all the trouble it takes to put them on. The vast array of intracellular protein functions achieved without attachment of N-linked oligosaccharides bears this out. While recognizing that attachment of sugars to proteins affects their intrinsic activities, one cannot help but feel that the sugars must be put there to serve some general purpose. The tangential effects on enzymatic or other activities have been exploited to various degrees in specific instances, but what is the general purpose? Recent results Minireview

Multivalent ligand binding by serum mannose-binding protein

The serum-type mannose-binding protein (MBP) is a defense molecule that has carbohydrate-dependent bactericidal effects. It shares with mammalian and chicken hepatic lectins similarity in the primary structure of the carbohydrate-recognition domain, as well as the ligand-binding mode: a high affinity (KD approximately nM) is generated by clustering of approximately 30 terminal target sugar residues on a macromolecule, such as bovine serum albumin, although the individual monosaccharides have low affinity (KD 0.1-1 mM). On the other hand, MBP does not manifest any significant affinity enhancement toward small, di- and trivalent ligands, in contrast to the hepatic lectins whose affinity toward divalent ligands of comparable structures increased from 100- to 1000-fold. Such differences may be explained on the basis of different subunit organization between the hepatic lectins and MBP.

Mechanism of N-acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain.

The mammalian hepatic asialoglycoprotein receptor, a member of the C-type animal lectin family, displays preferential binding to N-acetylgalactosamine compared with galactose. The structural basis for selective binding toN-acetylgalactosamine has been investigated. Regions of the carbohydrate-recognition domain of the receptor believed to be important in preferential binding to N-acetylgalactosamine have been inserted into the homologous carbohydrate-recognition domain of a mannose-binding protein mutant that was previously altered to bind galactose. Introduction of a single histidine residue corresponding to residue 256 of the hepatic asialoglycoprotein receptor was found to cause a 14-fold increase in the relative affinity forN-acetylgalactosamine compared with galactose. The relative ability of various acyl derivatives of galactosamine to compete for binding to this modified carbohydrate-recognition domain suggest that it is a good model for the natural N-acetylgalactosamine binding site of the asialoglycoprotein receptor. Crystallographic analysis of this mutant carbohydrate-recognition domain in complex withN-acetylgalactosamine reveals a direct interaction between the inserted histidine residue and the methyl group of theN-acetyl substituent of the sugar. Evidence for the role of the side chain at position 208 of the receptor in positioning this key histidine residue was obtained from structural analysis and mutagenesis experiments. The corresponding serine residue in the modified carbohydrate-recognition domain of mannose-binding protein forms a hydrogen bond to the imidazole side chain. When this serine residue is changed to valine, loss in selectivity forN-acetylgalactosamine is observed. The structure of this mutant reveals that the β-branched valine side chain interacts directly with the histidine side chain, resulting in an altered imidazole ring orientation.

Organization of the gene encoding the human macrophage mannose receptor (mrc1)

The gene for the human macrophage mannose receptor (MRC1) has been characterized by isolation of clones covering the entire coding region. Sequence analysis reveals that the gene is divided into 30 exons. The first three exons encode the signal sequence, the NH2-terminal cysteine-rich domain, and the fibronectin type II repeat, while the final exon encodes the transmembrane anchor and the cytoplasmic tail. The intervening 26 exons encode the eight carbohydrate-recognition domains and intervening spacer elements. However, no simple correlation between intron boundaries and functional carbohydrate-recognition domains is apparent. The pattern of intron positions as well as comparison of the sequences of the carbohydrate-recognition domains suggests that the duplication of these domains was an evolutionarily ancient event.