Marianne Olsen

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.

Characterization of Soluble Neural Cell Adhesion Molecule in Rat Brain, CSF, and Plasma

Abstract: The polypeptide composition and glycosylation of soluble isoforms of neural cell adhesion molecule (NCAM) in developing rat brain, CSF, and plasma were characterized. Soluble NCAM in rat brain consisted of several glycosylated isoforms. The degree of glycosylation was developmentally regulated. After desialylation, four polypeptides of Mr values of ∼ 190,000 (sl), 135,000 (s2), 115,000 (s3), and 110,000 (s4) were observed. Polypeptides si, s2, and s3 were also present in CSF, whereas only s3 and s4 were observed in plasma. Treatment of soluble brain NCAM with N‐glycosidase F, which removes N‐linked carbohydrates, produced polypeptides of Mr values of ∼ 190,000, 125,000, and 108,000–97,000. The monoclonal antibody OB11, which recognizes an epitope on the cytoplasmic part of transmembrane forms of NCAM, did not react with any of the soluble isoforms. Purified soluble NCAM, consisting mainly of s3, contained an N‐terminal sequence identical to that of membrane‐associated NCAM. Gel nitration of s3 indicated that it was present as a dimer under the chosen conditions. NCAM‐expressing glioma cells adhered specifically to immobilized soluble NCAM. This implies that functionally significant soluble forms of NCAM are present in the extracellular fluid.

Identification of NCAM-binding peptides promoting neurite outgrowth via a heterotrimeric G-protein-coupled pathway

A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I–F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted ENFIN2 and ENFIN11) were confirmed to bind to F3I–F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src‐related non‐receptor tyrosine kinase Fyn, and heterotrimeric G‐proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G‐proteins. Neurite outgrowth induced by trans‐homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2‐ and ENFIN11‐induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM‐dependent manner through G‐protein‐coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAM‐mediated signaling.

Expression of NCAM mRNA and polypeptides in aging rat brain

In aging brain, degenerative as well as compensatory regenerative processes are believed to occur. The neural cell adhesion molecule NCAM is involved in developmental and regenerative processes in the brain. However, the role of NCAM in aging brain has not been characterized. In this study, the expression of NCAM mRNAs and polypeptides was investigated in aging rat brain. The 7.4 and 6.7 kb NCAM mRNAs were selectively downregulated during postnatal development, and the 5.2 and 2.9 kb NCAM mRNAs were upregulated. However, from postnatal day 40 to old age no change in NCAM mRNA classes was observed. The fraction of NCAM mRNA containing the VASE exon increased postnatally but remained stable during adult life. VASE, which is believed to modulate the binding capacity, seemed to be relatively more abundant in the 7.4 and 6.7 kb NCAM mRNAs, encoding transmembrane NCAM forms, than in the 5.2 and 2.9 kb NCAM mRNAs, coding for glycosyl phosphatidylinositol (GPI) linked NCAM. Conversely, insertion of exons a and AAG between exons 12 and 13, a region containing two fibronectin type III repeats, seemed to be more pronounced in 5.2 and 2.9 kb NCAM mRNAs than in the 7.4 and 6.7 kb mRNAs. During postnatal development an increase in the fraction of 6.7 kb NCAM mRNA containing the exons a and AAG was observed. However, during aging the fraction of NCAM mRNAs containing this exon combination seemed constant. At the protein level, NCAM‐A was downregulated both during development and aging. No changes were observed during aging in the composition of soluble NCAM forms in the brain, cerebrospinal fluid or blood plasma. The amount of NCAM in rat brain decreased during postnatal development, but remained at a constant level from postnatal day 40 to old age.

Novel avidin and streptavidin binding sequences found in synthetic peptide libraries

Synthetic resin‐bound peptide libraries made of protein l‐amino acids have been synthesized. During screening of the libraries, peptides that bind to avidin have been identified containing a novel motif with two histidines separated by one residue. A sub‐library was synthesized and screened, and new critical residues appeared surrounding the two histidines. Additionally peptide libraries made of d‐amino acids have been screened with avidin and streptavidin and novel motifs have been found.

The ability to re-express polysialylated NCAM in soleus muscle after denervation is reduced in aged rats compared to young adult rats

The neural cell‐adhesion molecule, NCAM, contains an unusual homopolymer of sialic acid units, polysialic acid. This carbohydrate seems to be involved in neurite outgrowth, bundling and branching, processes which are important during reinnervation. In aged rats, reinnervation of denervated muscle fibres is incomplete. In this study, age‐related changes in the degree of polysialylation of NCAM re‐expressed after denervation were examined using a monoclonal antibody recognizing polysialic acid and a polyclonal antibody recognizing NCAM. The results show that, after denervation, the degree of polysialylation on NCAM was clearly reduced in rat soleus muscle of aged, compared to young, adult rats. This age‐related change in expression of polysialic acid probably influences the reinnervation process in aged muscle.

Erratum: Erratum to 'Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides”

On p. 1000 of the October 1999 issue, “Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides” by Lars C.B. Rønn, Marianne Olsen, Søren Østergaard, Vladislav Kiselyov, Vladimir Berezin, Marie T. Mortensen, Mathilde H. Lerche, Peter H. Jensen, Vladislav Soroka, Jane L. Saffell, Patrick Doherty, Flemming M. Poulsen, Elisabeth Bock, and Arne Holm, author Jane Saffells name was misspelled.