The online version of this article (https://doi.org/10.1186/s13024-018-0279-z) contains supplementary material, which is available to authorized users.
Neurogranin (Ng) is a small 7.6 kDa postsynaptic protein that has been detected at elevated concentrations in cerebrospinal fluid (CSF) of patients with Alzheimer’s disease (AD), both as a full-length molecule and as fragments from its C-terminal half. Ng is involved in postsynaptic calcium (Ca) signal transduction and memory formation via binding to calmodulin in a Ca-dependent manner. The mechanism of Ng secretion from neurons to CSF is currently unknown, but enzymatic cleavage of Ng may be of relevance. Therefore, the aim of the study was to identify the enzymes responsible for the cleavage of Ng, yielding the Ng fragment pattern of C-terminal fragments detectable and increased in CSF of AD patients.
Fluorigenic quenched FRET probes containing sequences of Ng were utilized to identify Ng cleaving activities among enzymes known to have increased activity in AD and in chromatographically fractionated mouse brain extracts.
Human Calpain-1 and prolyl endopeptidase were identified as the candidate enzymes involved in the formation of endogenous Ng peptides present in CSF, cleaving mainly in the central region of Ng, and between amino acids 75_76 in the Ng sequence, respectively. The cleavage by Calpain-1 affects the IQ domain of Ng, which may deactivate or change the function of Ng in Ca2+/calmodulin -dependent signaling for synaptic plasticity. While shorter Ng fragments were readily cleaved in vitro by prolyl endopeptidase, the efficiency of cleavage on larger Ng fragments was much lower.
Calpain-1 and prolyl endopeptidase cleave Ng in the IQ domain and near the C-terminus, respectively, yielding specific fragments of Ng in CSF. These fragments may give clues to the roles of increased activities of these enzymes in the pathophysiology of AD, and provide possible targets for pharmacologic intervention.
Additional file 1: Top 30 hits from Mascot search in Swiss-Prot (mouse) using tryptic peptides from excised band of C-terminal Ng cleaving activity. (DOCX 37 kb) (XLSX 10 kb)
Kvartsberg H, Portelius E, Andreasson U, Brinkmalm G, Hellwig K, Lelental N, et al. Characterization of the postsynaptic protein neurogranin in paired cerebrospinal fluid and plasma samples from Alzheimer's disease patients and healthy controls. Alzheimers Res Ther. 2015;7:40. CrossRefPubMedPubMedCentral
Pak JH, Huang FL, Li J, Balschun D, Reymann KG, Chiang C, et al. Involvement of neurogranin in the modulation of calcium/calmodulin-dependent protein kinase II, synaptic plasticity, and spatial learning: a study with knockout mice. Proc Natl Acad Sci U S A. 2000;97(21):11232–7. CrossRefPubMedPubMedCentral
Myöhänen TT, García-Horsman JA, Tenorio-Laranga J, Männistö PT. Issues about the physiological functions of prolyl oligopeptidase based on its discordant spatial association with substrates and inconsistencies among mRNA, protein levels, and enzymatic activity. J Histochem Cytochem. 2009;57(9):831–48. CrossRefPubMedPubMedCentral
- Alzheimer-associated cerebrospinal fluid fragments of neurogranin are generated by Calpain-1 and prolyl endopeptidase
Faisal Hayat Nazir
- BioMed Central