Identification of galactosyltransferase-i interaction surface by site directed mutagenesis and i̇n vi̇vo FRET (förster resonance energy transfer) analysis

Glycosylation is one of the most known post translational modification of proteins and lipids. Proper glycosylation is essential for many biological events such as cell recognition, immunological activity, cell signalling and biological diversity. Glycan branching and sugar addition are the backbone of glycosylation. The regulation of glycan branching is under control of glycosyltransferase enzymes. It is known that any defects of these enzymes leads to severe consequences for all organisms. Yet, the working mechanism and details of glycosyltransferases in both health and diseases remain unclear. Since 1960s, many of articles has been published that glycosyltransferases may form homomeric and heteromeric complexes with each other. The recent developments in bioinformatics, molecular biology and cell physiology, let us to know much more information about protein – protein interactions. The aim of this study is, to find out Beta 1-4 Galactosyltransferase-I enzyme interacting surface by using site directed mutagenesis and FRET technique. Therefore, we used ClusPro web server to predict protein – protein docking positions and relevant amino acids. After that, candidate amino acids (282 Try, 283 Val, 284 Glu, 359 Ile) which they have potentially responsible for homomeric interaction have been mutated into glycine and aspartic acid via using Agilent Site-Directed Mutagenesis Kit. The mutant cDNAs are inserted mCherry and mVenus FRET plasmid contructs then transfected to Cos-7 mammalian cell line. The localization of mutant enzymes confirmed by LSM confocal microscopy. FRET measurements performed in Operetta, PerkinElmer. As result, 282nd, 283rd and 359th regions might be possibly responsible for interacting surface. According to FRET ratios, particularly 283rd region valine could be a good interaction point between two GalT-1 homomers.