Myelin basic protein, an autoantigen in multiple sclerosis, is selectively processed by human trypsin 4

Medveczky, Peter G.; Antal, József; Patthy, András; Kékesi, Katalin A.; Juhász, Gábor; Szilágyi, László; Gráf, László

doi:10.1016/j.febslet.2005.12.067

Cited by 40 publications

(36 citation statements)

References 25 publications

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“…This phosphorylated form is associated with non-compact myelin and detergent-insoluble membrane microdomains ("lipid rafts") [69,73,78,79]. Phosphorylation protects the protein from proteases such as trypsin [80], and has potential structure-stabilizing effects [74,81,82]. The MAPK phosphorylation of MBP decreases its ability to bundle actin filaments and to bind actin filaments, microtubules, and the SH3-domain of Fyn to a lipid membrane [26,34,83].…”

Section: Discussionmentioning

confidence: 99%

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

et al. 2012

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The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multifunctional, having numerous protein-protein interactions with Ca 2+ -calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct 'membrane-ruffled' regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domaincontaining proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. CIHR Author ManuscriptCIHR Author Manuscript CIHR Author ManuscriptPreviously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.

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Section: Discussionmentioning

confidence: 99%

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

et al. 2012

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“…1). Human trypsin 4 has recently been shown to selectively process myelin basic protein in vitro, the most abundant membrane protein in the central nervous system (10). Upon proteolytic cleavage, it generates a peptide fragment comprising the sequence that is recognized by major antibodies found in patients with multiple sclerosis.…”

Section: From the Department Of Biochemistry Eötvös Loránd Universitmentioning

confidence: 99%

Thermodynamic Analysis Reveals Structural Rearrangement during the Acylation Step in Human Trypsin 4 on 4-Methylumbelliferyl 4-Guanidinobenzoate Substrate Analogue

Toth

Gombos

Simon

et al. 2006

Journal of Biological Chemistry

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Human trypsin 4 is an unconventional serine protease that possesses an arginine at position 193 in place of the highly conserved glycine. Although this single amino acid substitution does not affect steady-state activity on small synthetic substrates, it has dramatic effects on zymogen activation, interaction with canonical inhibitors, and substrate specificity toward macromolecular substrates. To study the effect of a non-glycine residue at position 193 on the mechanism of the individual enzymatic reaction steps, we expressed wild type human trypsin 4 and its R193G mutant. 4-Methylumbelliferyl 4-guanidinobenzoate has been chosen as a substrate analogue, where deacylation is rate-limiting, and transient kinetic methods were used to monitor the reactions. This experimental system allows for the separation of the individual reaction steps during substrate hydrolysis and the determination of their rate constants dependably. We suggest a refined model for the reaction mechanism, in which acylation is preceded by the reversible formation of the first tetrahedral intermediate. Furthermore, the thermodynamics of these steps were also investigated. The formation of the first tetrahedral intermediate is highly exothermic and accompanied by a large entropy decrease for the wild type enzyme, whereas the signs of the enthalpy and entropy changes are opposite and smaller for the R193G mutant. This difference in the energetic profiles indicates much more extended structural and/or dynamic rearrangements in the equilibrium step of the first tetrahedral intermediate formation in wild type human trypsin 4 than in the R193G mutant enzyme, which may contribute to the biological function of this protease.Trypsin is a prototype of the S1 serine protease family, the largest group of proteases. The residues indispensable for its enzymatic activity, His-57, , are structurally conserved and are referred to collectively as the catalytic triad (1). These residues are located at the active site of the enzyme. As the first step of the catalytic cycle, the enzyme binds the substrate forming the non-covalent Michaelis complex. The hydroxyl oxygen of the catalytic serine makes a nucleophilic attack on the electron-deficient carbonyl carbon of the scissile bond, and a covalent bond is formed. The attacked carbon atom becomes tetrahedrally coordinated; thus this state is called the first tetrahedral intermediate. In the next step, this species breaks down to yield the C-terminal (first) product and the acyl-enzyme. The acylation is followed by deacylation, a mechanistically similar action, in which the acyl-ester bond is attacked by a water molecule that has previously been activated by 3).During the hydrolysis of the scissile bond, tetrahedral intermediates are developed that are stabilized via hydrogen bonding interactions, in which the amido groups of residues Gly-193 and Ser-195 act as H-donors and the developing oxyanion intermediate is the acceptor (4, 5). Despite glycine being the far most convenient amino acid at position 193 for optimal...

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“…Overexpression of trypsinogen IV in neurons of the mouse brain results in a remarkable overexpression of glial fibrillary acidic protein in astrocytes, suggesting a role for trypsin IV in astrocyte proliferation (19). Trypsin IV also degrades myelin basic protein, the autoantigen of multiple sclerosis, suggesting an involvement in human disease (20). Thus, trypsin IV/mesotrypsin and p23 are up-regulated and prematurely activated during inflammation and in tumors, where the sustained, inhibitor-resistant activity of these proteases could contribute to inflammation, tumor formation, and disease progression by mechanisms that remain to be discovered.…”

mentioning

confidence: 99%

Trypsin IV or Mesotrypsin and p23 Cleave Protease-activated Receptors 1 and 2 to Induce Inflammation and Hyperalgesia

Knecht

Cottrell

Amadesi

et al. 2007

Journal of Biological Chemistry

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Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR 2 ؊/؊ mice but maintained in PAR 1 ؊/؊ mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR 1 -and PAR 2 -dependent inflammation and PAR 2 -dependent hyperalgesia.

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Myelin basic protein, an autoantigen in multiple sclerosis, is selectively processed by human trypsin 4

Cited by 40 publications

References 25 publications

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

Thermodynamic Analysis Reveals Structural Rearrangement during the Acylation Step in Human Trypsin 4 on 4-Methylumbelliferyl 4-Guanidinobenzoate Substrate Analogue

Trypsin IV or Mesotrypsin and p23 Cleave Protease-activated Receptors 1 and 2 to Induce Inflammation and Hyperalgesia

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