Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme

Abstract

The sialyltranferase ST3Gal-V transfers a sialic acid to lactosylceramide. We investigated the role of each of the N-glycans modifying mouse ST3Gal-V (mST3Gal-V) by measuring the in vitro enzyme activity of Chinese hamster ovary (CHO) cells transfected with ST3Gal-V cDNA or its mutants. By examining mutants of mST3Gal-V, in which each asparagine was replaced with glutamine (N180Q, N224Q, N334Q), we determined that all three sites are N-glycosylated and that each N-glycan is required for enzyme activity. Despite their importance, N-glycosylation sites in ST3Gal-V are not conserved among species. Therefore, we considered whether the function in the activity that is performed in mST3Gal-V by the N-glycan could be substituted for by specific amino acid residues selected from the ST3Gal-V of other species or from related sialyltransferases (ST3Gal-I, -II, -III, and -IV), placed at or near the glycosylation sites. To this end, we constructed a series of interspecies mutants for mST3Gal-V, specifically, mST3Gal-V-H177D-N180S (medaka or tetraodon type), mST3Gal-V-N224K (human type), and mST3Gal-V-T336Q (zebrafish type). The ST3Gal-V activity of these mutants was quite similar to that of the wild-type enzyme. Thus, we have demonstrated here that the N-glycans on mST3Gal-V are required for activity but can be substituted for specific amino acid residues placed at or near the glycosylation sites. We named this method SUNGA (substitution of N-glycan functions in glycosyltransferases by specific amino acids). Furthermore, we verified that the ST3Gal-V mutant created using the SUNGA method maintains its high activity when expressed in Escherichia coli thereby establishing the usefulness of the SUNGA method in exploring the function of N-glycans in vivo.

Zebrafish and Mouse α2,3-Sialyltransferases Responsible for Synthesizing GM4 Ganglioside

“…1) Cultured cells, including various cancer cell lines, highly express sialidases such as NEU3, which cleaves GM3 (46) and possibly GM4, and thus we added 40 M NeuAc to the reaction mixture to inhibit the sialidase activity in the enzyme preparation if necessary, and the reaction products (GM3 and GM4) eventually increased compared with the experiment without inhibitors. It is not clear whether such sialidase inhibitors were used for the assay of ST3GalVs in previous experiments.…”

The Cytoplasmic Tail of GM3 Synthase Defines Its Subcellular Localization, Stability, and In Vivo Activity

“…Proteins were separated by SDS-PAGE and transferred to an Immobilon polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membrane was then incubated for 1 h with a 1:1000 dilution of anti-SAT-I antibodies that had been raised against GST or polyhistidine fusion proteins encompassing the COOH-terminal 55 aa (C-term; Uemura et al, 2006) or 267 aa (9129). After washing, the membrane was incubated for 1 h with a 1:5000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit IgG F(abЈ) 2 fragment (GE Healthcare Bio-Sciences, Piscataway, NJ).…”

“…Each SAT-I protein was detected as a broad band of 44.5-48 kDa ( Figure 4B). Because mouse SAT-I carries three N-glycans (Uemura et al, 2006), samples of integral membrane fractions were also treated with PNGase F before immunoblotting. Interestingly, the broad bands in mSAT-Ia-transfected cells shifted to 46, 40, and 36 kDa, whereas the broad band from mSAT-Ib-transfected cells resolved into bands at 40 and 36 kDa ( Figure 4B).…”

“…However, in the mSAT-Ia, hSAT-Ia-1 and -Ia-2, and hSAT-Ib-1 and -Ib-2 variants, a second or third AUG, M3, has been considered as the initiation codon, based on speculation that the context presenting M1 and M2 is insufficient for either to be recognized by the ribosome as an initiation codon (Ishii et al, 1998;Kapitonov et al, 1999;Kim et al, 2001). Although the exact translational sites remain undetermined, the translational product from M3 (M3-SAT-I) has been used in many studies (Giraudo et al, 2001;Giraudo and Maccioni, 2003a,b;Uemura et al, 2006;Uliana et al, 2006;D'Angelo et al, 2007). Because the catalytic domain of the SAT-I protein exits on the lumen side of Golgi apparatus and M3-SAT-I can produce GM3 in vivo (Ishii et al, 1998), little attention has been given to the significance of the position of the initiation codon; however, the position of the initiation codon would determine the length of the cytoplasmic region of a SAT-I protein.…”

Glycoinformatics approach for identifying target positions to inhibit initial binding of SARS-CoV-2 S1 protein to the host cell

“…As glycan structures typically exist in solution or on proteins, it is a big challenge to characterize the 3D structure of glycoproteins experimentally. However, computational structural biology allows us to generate 3D protein and glycoprotein modelling with high accuracy rate using all amino acid sequence [14][15][16]. Herein, we predicted Oglycosylation and phosphorylation positions using full amino acid sequence of S1 protein.…”