7 research outputs found

    Neurofascin 186 Is O-Mannosylated within and Outside of the Mucin Domain

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    Protein O-mannosylation is an important modification in mammals, and deficiencies thereof lead to a variety of severe phenotypes. Although it has already been shown that the amount of O-mannosyl glycans in brain is very high, only very few proteins have been identified as O-mannosylated. Additionally, the functions of the O-mannose-based glycans are still speculative and only investigated for α-dystroglycan. In a previous study a cis-located peptide was identified, which controls O-mannosylation in mammals. A BLAST search on the basis of this peptidic determinant identified other potential O-mannosylated proteins. Among these neurofascin was chosen for further analysis as a recombinant probe (mucin domain) and as an endogenous protein from mouse brain. Mass spectrometric data for both proteins confirmed that neurofascin186 is indeed O-mannosylated. Glycopeptide analysis by liquid chromatography-tandem mass spectrometry allowed for the identification of some of the O-mannosylation sites, which are not restricted to the mucin domain but were found also within N-terminal IgG and Fibronectin domains of the protein

    The Lecticans of Mammalian Brain Perineural Net Are O‑Mannosylated

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    O-Mannosylation is an important protein modification in brain. During the last years, a few mammalian proteins have been identified as targets of the protein-<i>O</i>-mannosyltransferases 1 and 2. However, these still cannot explain the high content of O-mannosyl glycans in brain and the strong brain involvement of congenital muscular dystrophies caused by POMT mutations (Walker-Warburg syndrome, dystroglycanopathies). By fractionating and analyzing the glycoproteome of mouse and calf brain lysates, we could show that proteins of the perineural net, the lecticans, are O-mannosylated, indicating that major components of neuronal extracellular matrix are O-mannosylated in mammalian brain. This finding corresponds with the high content of <i>O</i>-mannosyl glycans in brain as well as with the brain involvement of dystroglycanopathies. In contrast, the lectican neurocan is not O-mannosylated when recombinantly expressed in EBNA-293 cells, revealing the possibility of different control mechanisms for the initiation of O-mannosylation in different cell types

    Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems

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    <div><p>Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.</p></div

    Enhanced expression of eGFP.

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    <p>HEK293 cells were transiently transfected with eGFP alone (basic) or fused to different expression tags. (A) All proteins contained a signal peptide sequence for secretion. The eGFP signals were visualized by fluorescence microscopy. (B) To quantify the secreted eGFP, the supernatants from the HEK293 cells were excited with a laser at 488 nm and the emission signal was detected at 509 nm (top). Densitometric analysis of secreted eGFP from HEK293 cells by western blot analysis using a Strep-Tactin®-HRP conjugate to detect Strep II tagged eGFP fusion proteins (bottom). (C) Without signal peptide, eGFP was transiently expressed intracellularly with or without fused MBP. eGFP signals were visualized by fluorescence microscopy. (a.u.: arbitrary unit).</p

    Biophysical analysis of MBP as well as MBP fusion proteins and cell attachment studies.

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    <p>(A) Light scattering profile obtained for a 1.80 mg/mL solution of MBP (dotted line) and for a 3.28 mg/mL solution of MBP-netrin-4 delta (solid line) in the TBS buffer display the respective hydrodynamic radius. (B) Concentration dependence of the hydrodynamic radius of MBP (dotted line) and MBP-netrin-4 delta (solid line), respectively, deduced from the peaks of DLS profiles. (C-D) B16-F1 cells were allowed to adhere to surface coated with MBP-netrin-4 delta. (C) Dependence of cell attachment on the coating concentration of MBP-netrin-4 delta. (D) Cell attachment at E<sub>50</sub> coating concentration (MBP-netrin-4 delta) for different netrin-4 delta proteins (with and without tag).</p

    Global application of the MBP tag as an expression enhancing tag.

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    <p>(A) Protein expression levels in HEK293 cells of intracellular proteins (HSP90α and Gli1) with and without MBP. (B) Western blot analysis of transmembrane proteins (MEGF9 and MMP14) cloned into the basic and the MBP vector system. (C) Comparison of the expression levels of netrin-4 delta in the basic and the MBP vector transfected into COS-7, CHO-K1, and HEK293 cells. (D) The N-terminal laminin β2LN-LEa1-4 fragment and the respective R246Q mutant version causing congenital nephrotic syndrome were expressed and western blot analysis was performed.</p

    Expression analysis in HEK293 cells of commonly used expression tags.

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    <p>(A-D) The expression levels were compared by western blot analysis detecting the double Strep II tag, which is present as an N-terminal tag on all proteins. (A) Different short arm laminin chains and netrins were transiently expressed and analyzed by western blot. (B) Different expression tags were transiently expressed and visualized by western blot. (C) Netrin-1 delta was fused to different expression tags. (D) Expression of netrin-4 delta in different vector systems was analyzed by western blot analysis. (Isom.: modified SlyD; mFc: Fc part from the mouse IgG protein; mFc-opt: codon optimized Fc part from the mouse IgG protein; ser alb: serum albumin; MBP: maltose binding protein; basic: without fusion protein).</p
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