Characterisation of novel glycoprotein and glycopolymer ligands of human C-type lectins

Transmembrane proteins of the C-type lectin family are major carbohydrate-binding components of the immune system that are present in abundance on the surfaces of cells, such as macrophages and dendritic cells, and function as receptors. Previous screening data using human placental extracts suggested that the glycoprotein pregnancy-associated plasma protein-A1 (PAPP-A1) may be a natural ligand for the C-type lectin DC-SIGN. Interaction data using state of the art dip-and-read real-time biosensor technology show that PAPP-A1 interacts with DC-SIGN and DC-SIGNR with dissociation constants consistent with the physiologically relevant, low nanomolar range. PAPP-A1 is a large glycoprotein complex and a metalloproteinase that influences insulin-like growth factor (IGF) bioavailability by cleaving IGFbinding proteins (IGFBP) 4 and 5. PAPP-A1 is found in abundance in plasma during pregnancy in complex with its natural inhibitor, eosinophil major basic protein (proMBP). Moreover, studies have shown that in males and non-pregnant females, PAPP-A1 circulates at much lower levels and is positively associated with acute coronary syndromes. Notably, PAPP-A1 possesses a high density of glycosylation sites and subsequent glycoproteomic analysis of native PAPP-A1/proMBP, and also recombinant PAPP-A1 and proMBP, was carried out via nano-liquid chromatography mass spectrometry. This glycoproteomic study successfully characterised the N-linked oligosaccharide profile of these molecules, including Nglycan site assignment. Additional interaction analysis also showed that cutting-edge star-shaped glycopolymers bind to DC-SIGN with picomolar affinity. Overall, the work illustrates the importance of glycoproteins and glycopolymers as potential modifiers of the immune system and also as factors associated with understanding the mechanisms of vascular diseases such as pre-eclampsia and acute coronary syndromes. There is also emphasis on the power of new, combined biophysical technologies in uncovering novel glycomic networks