A structural role for glycosylation: lessons from the hp model.

BACKGROUND: Protein glycosylation, the covalent attachment of carbohydrates, is very common, but in many cases the biological function of glycosylation is not well understood. Recently, fluorescence energy transfer experiments have shown that glycosylation can strongly change the global conformational distributions of peptides. We intend to show the physical mechanism behind this structural effect using a theoretical model. RESULTS: The framework of the hp model of Dill and coworkers is used to describe peptides and their glycosylated counterparts. Conformations are completely enumerated and exact results are obtained for the effect of glycosylation. On glycosylation, the model peptides experience conformational changes similar to those seen in experiments. This effect is highly specific for the sequence of amino acids and also depends on the size of the glycan. Experimentally testable predictions are made for related peptides. CONCLUSIONS: Glycans can, by means of entropic contributions, modulate the free energy landscape of polypeptides and thereby specifically stabilize polypeptide conformations. With respect to glycoproteins, the results suggest that the loss of chain entropy during protein folding is partly balanced by an increase in carbohydrate entropy.

Studies on human porin: XIII. The type-1 VDAC 'porin 31HL' biotinylated at the plasmalemma of trypan blue excluding human B lymphocytes.

In 1989, we demonstrated for the first time the expression of the VDAC 'Porin 31HL' in the plasmalemma of human B lymphocytes, then giving first evidence of a multi-topological expression of VDAC. Meanwhile, data from this and other laboratories support our proposal of a multi-compartment distribution of the channel in mammalian tissues. Here, by biotinylation of plasmalemma-integrated proteins of proven living B lymphocytes, followed by two-dimensional electrophoresis, immuno- and streptavidin affinity blotting, we show that part of the channel molecules can be labelled at the outer membrane of the cells. Thus, by a relevant approach our results invalidate objections concerning putative cross-reactivity of anti-human Type-1 porin antibodies with non-VDAC proteins at the outer cell membrane.

Studies on human porin. XII. Eight monoclonal mouse anti-"porin 31HL" antibodies discriminate type 1 and type 2 mammalian porin channels/VDACs in western blotting and enzyme-linked immunosorbent assays.

Eight mouse monoclonal antibodies directed against the acetylated N-terminal part of the type 1 human VDAC Porin 31HL clearly discriminate type 1 and type 2 mammalian porin channels. This is shown by comparing synthetic N-terminal peptides of either channel type in Western dot blots or by ELISA. The data support the specificity of the anti-Porin 31HL antibodies and thus give further support to our recent observations on extramitochondrial expression of VDAC. In the plasmalemma of different mammalian cells VDAC forms part of an ubiquitous chloride channel complex, which in patch clamp measurements may figure as the outwardly rectifying depolarization-induced chloride channel that is affected in cystic fibrosis.

Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes and bovine skeletal muscle, reversibly binds adenosine triphosphate (ATP).

A new aspect of mammalian porin (mammalian VDAC = mammalian voltage-dependent anion channel) is presented: channel active VDAC binds adenosine triphosphate (ATP) in the absence of Ca2+. Channel active "Porin 31HL" or "Porin 31BM", enriched from crude membranes of human B lymphocytes or whole cell lysates of bovine skeletal muscle, respectively, was bound to a nine atoms spacer ATP-agarose at pH 7.4 or 5.0 and reeluted from the resin by 10 mM ATP disodium salt. Furthermore, channel active "Porin 31BM" was labelled by [32P]ATP in a 1:1 stoichiometric relation. Binding of ATP to human porin was confirmed by studying the interaction of the synthetic porin fragment Type-1/Ac-35, comprising the putative nucleotide binding site G Y G F G, with trinitrophenyl-ATP (TNT-ATP) by scanning fluorometry. Peptide/TNP-ATP complexes clearly show enhancement of fluorescence intensity and a spectral shift of the fluorescence maximum. In a control experiment, using a porin fragment lacking the putative nucleotide binding site, no change of fluorescence emission was observed. Further confirmation for ATP binding by human VDAC arose from an autoradiographic experimental approach: the porin fragment Type-1/Ac-35 could be labelled by [32P]ATP, while a second porin fragment ending immediately before the putative nucleotide binding site could not; nor could a synthetic non porin peptide.

Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes or bovine skeletal muscle, reversibly binds the stilbene-disulfonate group of the chloride channel blocker DIDS.

Two new aspects of mammalian porin are presented. First, by affinity chromatography we show that channel active human or bovine porin reversibly bind the stilbene-disulfonate group of the chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). The procedure is suitable for further purification of porin after enrichment by ion exchange chromatography and shows a yield of 24.3%. The data support our recent proposal that VDAC forms part of the ORDIC channel complex which is affected in cystic fibrosis. Second, a purification scheme for mammalian porin is given starting with direct solubilisation of ground bovine skeletal muscle to avoid breaking up tissue. About 130 mg of channel active "Porin 31BM" are enriched from 946 g muscle tissue. Concerning its apparent molecular mass, primary structure, channel activity, channel conductance and voltage dependence the molecule shows high similarity to human porin. "Porin 31BM" is furthermore labelled by antibodies raised against human B lymphocyte derived "Porin 31HL".