Detection of Myelin Basic Protein Isoforms by Organic Concentration

Neurotrauma, as in the case of traumatic brain injury, promotes protease over‐activation characterized by the select fragmentation of brain proteins. The resulting polypeptides are indicators of biochemical processes, which can be used to study post‐injury dynamics and may also be developed into biomarkers. To this end, we devised a novel mass spectrometry approach to characterize post‐injury calpain proteolytic processing of myelin basic protein (MBP), a biomarker of brain injury that denotes white matter damage and recovery. Our approach exceeds conventional immunological assays in its deconvolution of multiple protein isoforms, its absolute quantification of proteolytic fragments and its polypeptide selectivity. We quantified and characterized post‐injury proteolytic processing of all MBP isoforms identified in adult rat cortex. Further, the translation of calpain‐cleaved MBP into CSF was verified following brain injury. We ascertained that the exon‐6 sequence of MBP resulted in a characteristic shift in gel migration for intact and fragmented protein alike. We also found evidence for a second post‐TBI cleavage event within exon‐2 and for the dimerization of the post‐TBI 4.3 kDa fragment. Ultimately, the novel methodology described here can be used to study MBP dynamics and other similar proteolytic events of relevance to brain injury and other CNS processes.

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“…(2002). In earlier rat MBP studies, isobaric 17 kDa isoforms were indistinguishable (Määttä et al. 1997; Kruger et al.…”

Section: Resultsmentioning

confidence: 89%

“…Intact MBP dimerization was noted elsewhere (Boulias et al. 1995; Määttä et al. 1997), though an exclusive link with the exon‐5/7 tandem sequence was not established.…”

Section: Resultsmentioning

confidence: 99%

Proteolysis of multiple myelin basic protein isoforms after neurotrauma: characterization by mass spectrometry

Ottens

Golden

Bustamante

et al. 2007

Journal of Neurochemistry

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“…Myelin basic protein (MBP) was extracted and purified from frozen brains of mice according to the method described by Määttä et al (1997). In brief, 3.08 g brain tissues were homogenized at room temperature in 15 ml chloroform by four 30 s bursts at full speed, intervened by 30 s pauses to avoid excess heating.…”

Section: Extraction and Purification Of Myelin Basic Protein (Mbp)mentioning

confidence: 99%

Modulatory effect of celastrol on Th1/Th2 cytokines profile, TLR2 and CD3+ T-lymphocyte expression in a relapsing–remitting model of multiple sclerosis in rats

Abdin

Hasby

2014

European Journal of Pharmacology

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“…The ability of H 6 ‐PSKR1‐KD to phosphorylate other proteins was shown with the common substrate myelin basic protein (MBP), the phosphorylated protein with a size of about 21.5 kDa corresponds to monomeric MBP with a typical retained migration on the gel (Cheifetz and Moscarello, ). The phosphorylated proteins with a size of about 24 kDa and 40 kDa could correspond to the multimeric complexes of MBP that were reported previously (Määttä et al ., ). An invariant Lys residue in subdomain II is directly involved in the phosphotransfer reaction of kinases (Kamps and Sefton, ) (Figures S1 and 1a,b).…”

Section: Resultsmentioning

confidence: 97%

Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1

et al. 2014

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SUMMARYThe cell growth-promoting peptide phytosulfokine (PSK) is perceived by leucine-rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca 2+ sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto-and transphosphorylation. Replacement of a conserved lysine within the ATP-binding region by a glutamate resulted in the inhibition of auto-and transphosphorylation kinase activities. Expression of the kinase-inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth-inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM)-binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM-like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM-binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase-inactive receptor. We conclude that PSK signaling requires Ca 2+ /CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth-promoting signaling pathway(s).

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Detection of Myelin Basic Protein Isoforms by Organic Concentration

Cited by 29 publications

References 28 publications

Proteolysis of multiple myelin basic protein isoforms after neurotrauma: characterization by mass spectrometry

Proteolysis of multiple myelin basic protein isoforms after neurotrauma: characterization by mass spectrometry

Modulatory effect of celastrol on Th1/Th2 cytokines profile, TLR2 and CD3+ T-lymphocyte expression in a relapsing–remitting model of multiple sclerosis in rats

Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1

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