Alzheimer-associated cerebrospinal fluid fragments of neurogranin are generated by Calpain-1 and prolyl endopeptidase

Bruno Becker,Kaj Blennow,Martina Boström,Henrik Zetterberg,Faisal Hayat Nazir,Elena Camporesi,Marie Kalm,Kina Höglund,Maria Olsson,Erik Portelius,Hlin Kvartsberg,Gunnar Brinkmalm

doi:10.1186/s13024-018-0279-z

Abstract

BackgroundNeurogranin (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.MethodsFluorigenic 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.ResultsHuman 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.ConclusionsCalpain-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.

Highlights

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
Identification of calpain-1 as the enzyme cleaving Ng in the IQ domain Most of the Ng peptides identified in human CSF showed N-terminal endings between amino acid 41 and 49 and C-terminal endings between aa 75 and 78
Since the Myc-DDK tag on the fusion protein may have influenced the cleavage pattern on the Ng_Myc-DDK fusion protein, Ng was expressed in the Small ubiquitin-like modifier (SUMO) expression system, which allows the complete removal of expression vector-derived amino acids from the expressed Ng

Summary

Introduction

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. Ng has two known mutually exclusive intracellular binding partners, calmodulin (CaM) [15] and phosphatidic acid (PA) [16]. Essential for this binding is the IQ domain of Ng, so-called because of the presence of the amino acids isoleucine (I) and glutamine (Q). The main function for Ng appears to be to modulate CaM’s signal transduction pathways of CaM dependent enzymes to enhance synaptic plasticity [12] in long term potentiation (LTP)

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