Proteins destined for secretion, after folding and the initial modification in the endoplasmic reticulum (ER), are transported to the ERGIC and then to Golgi stacks, before being sorted into their final destinations. Collectively these proteins that move through the secretory pathway are known as cargo proteins. Some of the soluble cargo proteins require cargo receptors to be efficiently packaged into vesicles exiting the ER. Evidence for the presence of such receptors in mammals came from the studies of a genetic disorder called the combined deficiency of factor V (FV) and factor VIII (FVIII) or F5F8D. F5F8D patients have mutations in either of the two genes - LMAN1 or MCFD2. LMAN1 and MCFD2 form a receptor complex that facilitates the ER to Golgi transport of FV and FVIII.
Distinct binding sites for FV and FVIII exist on LMAN1 and MCFD2, but the specific location of these binding sites are not known. Ambiguity exists on the mechanism of cargo release by LMAN1 and MCFD2. Like FV and FVIII, other LMAN1 cargos (cathepsin C, Z and α-1 antitrypsin) are also glycosylated. Thus the sugar binding activity of carbohydrate recognition domain (CRD) of LMAN1 is presumably important for the recognition of the cargo glycoprotein.
Using biochemical approaches we determined the locations of sugar binding sites and their importance in cargo binding. We successfully purified the CRD of LMAN1 using a denaturation/refolding protocol. Using isothermal titration calorimetry (ITC) assay, we confirmed that our purified CRD is a functional protein by its ability to bind to 2 calcium ions. ITC assay also provided insight on the effect of pH on binding ability of CRD to calcium as well as revealed the binding affinity of CRD towards its ligand α1-2 mannobiose. In collaboration with Dr. Saurav Misra's lab, we solved the structure of mannobiose bound CRD which revealed key amino acids involved in ligand binding. Mutations of these amino acids in LMAN1 were constructed to reconfirm their importance in sugar binding in vivo. These biochemical studies will provide a useful insight into the mechanism of cargo binding in the ER and release in the ERGIC and Golgi.