Cumulative phylogenetic, sequence and structural analysis of Insulin superfamily proteins provide unique structure-function insights.

The insulin superfamily proteins (ISPs), in particular, insulin, IGFs and relaxin proteins are key modulators of animal physiology. They are known to have evolved from the same ancestral gene and have diverged into proteins with varied sequences and distinct functions, but maintain a similar structural architecture stabilized by highly conserved disulphide bridges. The recent surge of sequence data and the structures of these proteins prompted a need for a comprehensive analysis, which connects the evolution of these sequences (427 sequences) in the light of available functional and structural information including representative complex structures of ISPs with their cognate receptors. This study reveals (a) unusually high sequence conservation of IGFs (>90 % conservation in 184 sequences) and provides a possible structure-based rationale for such high sequence conservation; (b) provides an updated definition of the receptor-binding signature motif of the functionally diverse relaxin family members (c) provides a probable non-canonical C-peptide cleavage site in a few insulin sequences. The high conservation of IGFs appears to represent a classic case of resistance to sequence diversity exerted by physiologically important interactions with multiple partners. We also propose a probable mechanism for C-peptide cleavage in a few distinct insulin sequences and redefine the receptor-binding signature motif of the relaxin family. Lastly, we provide a basis for minimally modified insulin mutants with potential therapeutic application, inspired by concomitant changes observed in other insulin superfamily protein members supported by molecular dynamics simulation.

Synthesis and Evaluation of the Insulin-Albumin Conjugate with Prolonged Glycemic Control.

Engineering therapeutic proteins to improve their half-life so as to sustain physiologically relevant extended activity is the need of the hour in biopharmaceutical research. In this study, insulin and bovine serum albumin (BSA) were independently functionalized rationally and were later conjugated to prolong the half-life of insulin. The thiol functionalization of BSA with 2-imminothiolane in the ratio 1:20 yielded an average of 6-8 thiols/BSA, which then reacted with maleimide-functionalized insulin to form an insulin-albumin conjugate. The bioconjugate was purified by size exclusion chromatography, and the increase in size was confirmed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Bioconjugation resulted in a multi-fold increase in the hydrodynamic volume of the insulin-albumin conjugate as measured in DLS when compared to BSA. The glucose uptake assay with 3LT3-L1 cell lines was performed, and the mean fluorescence intensity (MFI) of 16.16 observed for the insulin-albumin conjugate was comparable to insulin (19.42). The blood glucose reducing capacity of the insulin-albumin conjugate in streptozotocin induced diabetic male Wistar rats was well maintained up to 72 h when compared to native insulin. Further, a three-fold increase in plasma insulin concentration was observed in bioconjugate treated animals as against insulin treated animals after 24 h of treatment using ELISA. The histological analysis of different organs of the bioconjugate treated rats indicated that it was non-toxic. This study has paved a way for further detailed studies on similar bioconjugates to develop next-generation biotherapeutics for treating diabetes.