13 research outputs found

    Proteomic Analysis of Tissue from α1,3-galactosyltransferase Knockout Mice Reveals That a Wide Variety of Proteins and Protein Fragments Change Expression Level

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    A barrier in a pig-to-man xenotransplantation is that the Galα1-3Galβ1-4GlcNAc-R carbohydrate (α-Gal epitope) expressed on pig endothelial cells reacts with naturally occurring antibodies in the recipient’s blood leading to rejection. Deletion of the α1,3-galactosyltransferase gene prevents the synthesis of the α-Gal epitope. Therefore, knockout models of the α1,3-galactosyltransferase gene are widely used to study xenotransplantation. We have performed proteomic studies on liver and pancreas tissues from wild type and α1,3-galactosyltransferase gene knockout mice. The tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis and liquid chromatography - tandem mass spectrometry. The analyses revealed that a wide variety of proteins and protein fragments are differentially expressed suggesting that knockout of the α1,3-galactosyltransferase gene affects the expression of several other genes

    Spot No. 2514 is a fragment of α-tubulin

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    <p>A) Spot 2514 migrates with a molecular mass below 40 kDa. The identified peptides from the spot are distributed in the N-terminal of α-tubulin and spot 2514 thus corresponds to a fragment of α-tubulin, since α-tubulin possesses a molecular mass around 50 kDa.</p

    GS1B4 staining of wt and α1,3-galactosyltransferase KO mouse tissues.

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    <p>The lectin stains the central vein (v) and the sinusoids are outlined in sections from the wt mouse liver (A) while there is no reaction in sections from the KO mouse liver (B). There is no staining of the blood vessels in pancreas from neither the wt mouse (C) nor from the KO mouse (D). The endocrine cells in islets of Langerhans (IL) are unstained while the exocrine cells in both the wt and the KO mouse show reaction. The green fluorescence reflects lectin staining and blue fluorescence reflects DAPI staining of the nuclei.</p

    Insulin analog with additional disulfide bond has increased stability and preserved activity

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    Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function
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