demonstration-heterologous-complexes-formed-golgi-resident-type-iii

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous ...

University of Wroclaw

Split luciferase complementation assay is one of the approaches enabling identification and analysis of protein-protein interactions in vivo. The NanoBiT technology is the most recent improvement of this strategy. Nucleotide sugar transporters and glycosyltransferases of the Golgi apparatus are the key players in glycosylation. Here we demonstrate the applicability of the NanoBiT system for studying homooligomerization of these proteins. We also report and discuss a novel heterologous interaction between UDP-galactose transporter and beta-1,4-galactosyltransferase 1.

Analysis of homologous and heterologous interactions between UDP-galactose transporter and beta-1,4-galactosyltransferase 1 using NanoBiT.

β1,4-galactosyltransferase 4 (B4GalT4) is one of seven B4GalTs that belong to CAZy glycosyltransferase family 7 and transfer galactose to growing sugar moieties of proteins, glycolipids, glycosaminoglycans as well as single sugar for lactose synthesis. Herein, we identify two asparagine-linked glycosylation sites in B4GalT4. We found that mutation of one site (Asn220) had greater impact on enzymatic activity while another (Asn335) on Golgi localization and presence of N-glycans at both sites is required for production of stable and enzymatically active protein and its secretion. Additionally, we confirm B4GalT4 involvement in synthesis of keratan sulfate (KS) by generating A375 B4GalT4 knock-out cell lines that show drastic decrease in the amount of KS proteoglycans and no significant structural changes in N- and O-glycans. We show that KS decrease in A375 cells deficient in B4GalT4 activity can be rescued by overproduction of either partially or fully glycosylated B4GalT4 but not with N-glycan-depleted B4GalT4 version.

Wojciech Wiertelak

Laboratory of Biochemistry,
Faculty of Biotechnology,
Laboratory of Biochemistry, Faculty of Biotechnology

University of Wroclaw

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Wojciech Wiertelak

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University of Wroclaw

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Laboratory of Biochemistry,
Faculty of Biotechnology,
Laboratory of Biochemistry, Faculty of Biotechnology