Generation and intracellular trafficking of a polysialic acid-carrying fragment of the neural cell adhesion molecule NCAM to the cell nucleus

Nina Westphal,Melitta Schachner,Gabriele Loers,David Lutz,Ralf Kleene,Thomas Theis

doi:10.1038/s41598-017-09468-8

Abstract

Polysialic acid (PSA) and its major protein carrier, the neural cell adhesion molecule NCAM, play important roles in many nervous system functions during development and in adulthood. Here, we show that a PSA-carrying NCAM fragment is generated at the plasma membrane by matrix metalloproteases and transferred to the cell nucleus via endosomes and the cytoplasm. Generation and nuclear import of this fragment in cultured cerebellar neurons is induced by a function-triggering NCAM antibody and a peptide comprising the effector domain (ED) of myristoylated alanine-rich C kinase substrate (MARCKS) which interacts with PSA within the plane of the plasma membrane. These treatments lead to activation of the fibroblast growth factor (FGF) receptor, phospholipase C (PLC), protein kinase C (PKC) and phosphoinositide-3-kinase (PI3K), and subsequently to phosphorylation of MARCKS. Moreover, the NCAM antibody triggers calmodulin-dependent activation of nitric oxide synthase, nitric oxide (NO) production, NO-dependent S-nitrosylation of matrix metalloprotease 9 (MMP9) as well as activation of matrix metalloprotease 2 (MMP2) and MMP9, whereas the ED peptide activates phospholipase D (PLD) and MMP2, but not MMP9. These results indicate that the nuclear PSA-carrying NCAM fragment is generated by distinct and functionally defined signal transducing mechanisms.

Highlights

In mammals, NCAM is the predominant carrier of Polysialic acid (PSA), a polymer of α2,8-linked sialic acid monomers
To substantiate that the nuclear PSA is attached to the NCAM fragment that is generated by matrix metalloprotease 2 (MMP2)- and matrix metalloprotease 9 (MMP9)-mediated cleavage, we performed immunoprecipitations with PSA antibody using the nuclear fractions from cultured cerebellar neurons after treatment with NCAM-Fc in the absence and presence of MMP2 or MMP9 inhibitors and treated the immunoprecipitates with peptide-N-glycosidase F to remove N-glycans including PSA
Levels of the major N-deglycosylated NCAM fragment were higher in immunoprecipitates from neurons stimulated with NCAM-Fc in the absence of inhibitors than in those treated with Fc, whereas levels of the N-deglycosylated NCAM fragment were not increased upon NCAM antibody stimulation in the presence of the MMP2- or MMP9-specific inhibitors (Fig. 1e)

Summary

Introduction

NCAM is the predominant carrier of PSA, a polymer of α2,8-linked sialic acid monomers. Proteolytic processing of NCAM by different proteases at the cell surface modulates cell interactions, and the resulting fragments influence several cellular events, such as neurite outgrowth[12,13,14,15,16,17]. We had found that PSA-lacking and -carrying proteolytic NCAM fragments comprising the intracellular and transmembrane domains as well as part of the extracellular domain enter the cell nucleus after their generation at the plasma membrane[18, 19]. The PSA-lacking transmembrane NCAM fragment is generated by a serine protease at the plasma membrane upon stimulation of cultured cerebellar neurons or NCAM-expressing transfected CHO cells with surrogate ligands, e.g. function-triggering NCAM antibody, and reaches the cell nucleus via the endoplasmic reticulum and cytoplasm in a calmodulin-dependent manner[18]. Our results show that generation and nuclear import of the PSA-carrying and PSA-lacking NCAM fragments are mediated by different mechanisms

Methods

Results

Conclusion