Dataset Information

The human colonic thiamine pyrophosphate transporter (hTPPT) is a glycoprotein and N-linked glycosylation is important for its function.

ABSTRACT: The recently identified human thiamine pyrophosphate transporter (hTPPT; product of the SLC44A4 gene) is responsible for absorption of the microbiota-generated TPP in the large intestine. The hTPPT is highly expressed in the colon, but not in other regions of the intestinal tract and is localized exclusively at the apical membrane domain of epithelia. The hTPPT protein is predicted to have multiple TM domains with a number of putative N-glycosylation sites, but it is not known if the protein is actually glycosylated, and if so at which site, and their role in the functionality of the transporter. Using several approaches including inhibiting de novo N-glycosylation in human colonic epithelial NCM460 cells with tunicamycin as well as enzymatic de-glycosylation, we show that the hTPPT protein is, indeed, a glycoprotein. Glycosylation of hTPPT was shown, by mean of site-directed mutagenesis, to occur at Asn(69), Asn(155), Asn(197), Asn(393), and Asn(416). However, only N-glycosylation at Asn(69), Asn(155), and Asn(393) appeared to be important for transporter functionality possibly through an effect on protein conformation and/or interaction with its ligand (but not through changes in expression at the cell membrane as determined by live cell confocal imaging). Results of this study showed, for the first time, that the hTPPT is glycosylated and that N-linked glycosylation occurs at multiple sites with some of them being important for function. The results also provide an indirect support for a membrane topology for hTPPT with 10 transmembrane domains as predicted by the TMHMM transmembrane helixes prediction program.

SUBMITTER: Nabokina SM

PROVIDER: S-EPMC4779673 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The human colonic thiamine pyrophosphate transporter (hTPPT) is a glycoprotein and N-linked glycosylation is important for its function.

Nabokina Svetlana M SM Subramanian Veedamali S VS Said Hamid M HM

Biochimica et biophysica acta 20160130 4

The recently identified human thiamine pyrophosphate transporter (hTPPT; product of the SLC44A4 gene) is responsible for absorption of the microbiota-generated TPP in the large intestine. The hTPPT is highly expressed in the colon, but not in other regions of the intestinal tract and is localized exclusively at the apical membrane domain of epithelia. The hTPPT protein is predicted to have multiple TM domains with a number of putative N-glycosylation sites, but it is not known if the protein is ...[more]

PMID: 26828122

Similar Datasets

Project description:The CRT (creatine transporter) is a member of the Na+- and Cl--dependent neurotransmitter transporter family and is responsible for the import of creatine into cells, and thus is important for cellular energy metabolism. We established for CRT an expression system in HEK-293 cells that allowed biochemical, immunological and functional analysis of CRT wild-type and glycosylation-deficient mutants. Analysis of HA (haemagglutinin)-tagged CRT-NN (wild-type rat CRT with an HA-tag at the C-terminus) revealed several monomeric immunoreactive species with apparent molecular masses of 58, 48 and 43 kDa. The 58 kDa species was shown to be plasma-membrane-resident by EndoHf (endoglycosidase Hf) and PNGase F (peptide N-glycosidase F) treatments and represents fully glycosylated CRT, whereas the 48 kDa and 43 kDa species were glycosylation intermediates and non-glycosylated CRT respectively. Glycosylation-deficient mutants (Asn192Asp, Asn197Asp and Asn192Asp/Asn197Asp) showed altered electrophoretic mobility, indicating that CRT is indeed N-glycosylated. In addition, a prominent CRT band in the range of 75-91 kDa was also detected. Pharmacological inhibition of N-linked glycosylation by tunicamycin in CRT-NN-expressing cells gave a similar reduction in molecular mass, corroborating the finding that Asn192 and Asn197 are major N-glycosylation sites in CRT. Although the apparent Km was not significantly affected in glycosylation-deficient mutants compared with CRT-NN, we measured reduced Vmax values for all mutants (21-28% residual activity), and 51% residual activity after enzymatic deglycosylation of surface proteins in intact CRT-NN cells by PNGase F. Moreover, immunocytochemical analysis of CRT-NN- and CRT-DD-expressing cells (where CRT-DD represents a non-glycosylated double mutant of CRT, i.e. Asn192Asp/Asn197Asp) showed a lower abundance of CRT-DD in the plasma membrane. Taken together, our results suggest that plasma-membrane CRT is glycosylated and has an apparent monomer molecular mass of 58 kDa. Furthermore, N-linked glycosylation is neither exclusively important for the function of CRT nor for surface trafficking, but affects both processes. These findings may have relevance for closely related neurotransmitter transporter family members.

Project description:Many viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reduced in vitro fusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in the Varicellovirus genus, resulted in enhanced in vitro fusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles.IMPORTANCE Herpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide fusion protein gB depends on the presence of the gH/gL complex for activation. Viral envelope glycoproteins, such as gH, usually contain N-glycans, which can have a strong impact on their folding, transport, and functions. Here, we systematically analyzed the functional relevance of all five predicted N-linked glycosylation sites in the alphaherpesvirus pseudorabies virus (PrV) gH. Despite the fact that mutation of specific sites affected gH transport, in vitro fusion activity, and cell-to-cell spread and resulted in delayed penetration kinetics, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Thus, our results demonstrate a modulatory but nonessential role of N-glycans for gH function.

Dataset Information

The human colonic thiamine pyrophosphate transporter (hTPPT) is a glycoprotein and N-linked glycosylation is important for its function.

Publications

The human colonic thiamine pyrophosphate transporter (hTPPT) is a glycoprotein and N-linked glycosylation is important for its function.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets