My research interest is in structure-function analysis of proteins, particularly of carbohydrate-recognition proteins involved in the innate immune response against pathogens and in glycoprotein homeostasis. The work has focussed on understanding molecular mechanisms involved in selective recognition of glycoproteins and cell surface carbohydrates and the biological functions mediated by carbohydrate recognition.
Molecular characterization of the mannose receptor
My early career focussed on two proteins, the mannose receptor and serum mannose-binding protein. The mannose receptor of liver endothelial cells and macrophages clears proteins such as lysosomal enzymes that bear high mannose oligosaccharides and are released during pathological events and plays a role in innate immunity against micro-organisms. As a graduate student I studied the cell biology and kinetics of clearance of glycoproteins by the hepatic mannose receptor and characterized soluble mannose-binding proteins from human serum. I cloned and sequenced the gene for the major human serum mannose-binding protein, which mediates complement-dependent killing of micro-organisms. Later, I cloned the cDNA for the human mannose receptor and found it to have an unusual multidomain structure including eight C-type carbohydrate-recognition domains. This arrangement of domains has since been found in three other endocytic receptors, making the mannose receptor the prototype for a family of multi-domain receptors. Subsequently I characterized the roles of the various domains of the receptor with the goal of gaining a molecular understanding of how the receptor binds and internalizes pathogens and harmful glycoproteins. Using a combination of biochemical, biophysical, cell and molecular biological approaches, we showed that several of the C-type carbohydrate-recognition domains are involved in binding and internalization of mannose-terminated glycoconjugates. We characterized the mechanism of calcium and sugar binding by the receptor, determined the crystal structure of the principle ligand-binding carbohydrate-recognition domain, in collaboration with Bill Weis at Stanford, and determined a mechanism by which the receptor can release its ligands in the endosomes. We showed that the mannose receptor has an additional function in binding of collagen through the fibronectin type II domain. We helped apply methods developed in studying the mannose receptor to other members of the "mannose receptor family", such as Endo-180. We have provided cDNA clones and cell lines expressing the mannose receptor to many groups world-wide to facilitate their studies on biological functions of the mannose receptor.
Molecular characterization of other C-type lectins
C-type lectins identified from genome sequence data are now the subject of much of our work. Examples include langerin, a receptor specific to Langerhans cells, and the scavenger receptor C-type lectin of endothelial cells. For langerin, we determined oligomeric structure, ligand-binding specificity, demonstrated the ability to mediate endocytosis of glycoproteins and recently solved the crystal structure of the trimeric extracellular region of the receptor, showing the disposition of the ligand-binding sites. We have shown that amino-acid changes due to polymorphisms in the langerin gene affect stability and ligand-binding by langerin, a finding that is important in understanding susceptibility to infectious organisms, such as HIV, that interact with langerin. For the scavenger receptor C-type lectin, in addition to biochemical characterization of the protein we have demonstrated high specificity for binding to the Lewis-X trisaccharide and determined the mechanism of this interaction by solving the crystal structure of the carbohydrate-recognition domain in complex with Lewis-X. The specificity of this receptor for a single endogenous oligosaccharide suggests a role in cell adhesion or clearance of specific glycoproteins. We have also contributed to molecular characterization of several other proteins involved in binding of viruses and other pathogens including the dendritic cell receptor DC-SIGN and the endothelial cell receptors DC-SIGNR and LSECtin and have recently identified new C-type lectins in the human genome.
Communication to a wider audience
To make the field of glycobiology accessible to undergraduates, I co-authored the first textbook in this area. Introduction to Glycobiology is now going into its 3rd edition and has been translated into Japanese, Chinese and Korean.