Dataset Information

Glycosylation increases the thermostability of human aquaporin 10 protein.

ABSTRACT: Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.

SUBMITTER: Oberg F

PROVIDER: S-EPMC3173105 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Glycosylation increases the thermostability of human aquaporin 10 protein.

Öberg Fredrik F Sjöhamn Jennie J Fischer Gerhard G Moberg Andreas A Pedersen Anders A Neutze Richard R Hedfalk Kristina K

The Journal of biological chemistry 20110706 36

Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylatio ...[more]

PMID: 21733844

Similar Datasets

Project description:BackgroundGlycerol outflow from adipocytes has been considered for a decade to be mediated by aquaporin-7, an aquaglyceroporin highly expressed in the adipose tissue. Its involvement in glycerol metabolism has been widely studied also in humans. Recent studies in different aquaporin-7 KO mice models pose two different questions 1) the exact localization of aquaporin-7 in human white adipose tissue; 2) the existence of other aquaglyceroporins that work with aquaporin-7 to guarantee glycerol efflux and thus a normal adiposity in humans. To this purpose we investigated the expression, the localization and the functioning of aquaglyceroporin-10 in subcutaneous white adipose tissue, in isolated and cultured differentiated adipocytes.Methodology/principal findingsAquaporin-7 and -10 were expressed in the white adipose tissue both at mRNA and at protein level. Immunofluorescence revealed aquaporin-7 and -10 labelling in the human adipose tissue both to the plasma membrane and to a thin rim of cytoplasm of adipocytes. Aquaporin-7, but not aquaporin-10, colocalized with the endothelial marker CD34. Human cultured differentiated adipocytes showed an aquaporin-7 and -10 labelling mainly in the cytoplasm and in the lipid droplets with insulin reinforcing the lipid droplets staining and isoproterenol inducing its translocation to the plasma membrane compartment. Water and glycerol permeability measurements using adipocytes and adipose membrane vesicles confirmed the presence of functioning aquaglyceroporins. Aquaporin-10 silencing in human differentiated adipocytes resulted in a 50% decrease of glycerol and osmotic water permeability.Conclusions/significanceThe results indicate that aquaporin-7, differently from mice, is present in both adipocyte and capillary plasma membranes of human adipose tissue. Aquaporin-10, on the contrary, is expressed exclusively in the adipocytes. The expression of two aquaglyceroporins in human adipose tissue is particularly important for the maintenance of normal or low glycerol contents inside the adipocyte, thus protecting humans from obesity.

Project description:AimB-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated.MethodsEndogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma model was employed to evaluate in vivo tumor growth and angiogenesis.ResultsResults demonstrated that Bcl2L10 acts as an inducer of in vitro cell migration, invasion, and VM, while in vitro cell proliferation, in vivo tumor growth, as well as colony formation properties were not affected. Dissecting different signaling pathways, it was found that Bcl2L10 positively affects the phosphorylation of extracellular-signal-regulated kinase (ERK) and the expression of markers of cell invasion, such as urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinases (MMPs). Of note, Bcl2L10-dependent in vitro migration, invasion, and VM are linked to uPAR. Bcl2L10 also negatively regulates the intracellular calcium level. Finally, reduced invasion capability in 3D spheroid invasion assay of melanoma cells transiently overexpressing Bcl2L10 was observed after treatment with inhibitors of MMPs and uPAR.ConclusionsOverall, data reported in this paper provide evidence supporting a positive role of Bcl2L10 in melanoma aggressive features.

Dataset Information

Glycosylation increases the thermostability of human aquaporin 10 protein.

Publications

Glycosylation increases the thermostability of human aquaporin 10 protein.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets