The Omgp Different Domains In Neurite Growth Inhibition In The Role

Oligodendrocyte Myeline Glycoprotein (OMgp) is a glycosylphosphatidylinositol-linked glycoprotein mainly expressed on the external surface of oligodendrocytes. Interestingly, the OMgp gene is embedded within an intron of the large neurofibromatosis type 1 (NF1) gene and on the opposite strand. The distribution analysis of OMgp mRNA in central nerves system (CNS) showed that they expressed in many classes of neurons, but their function remain unclear. Wang et al. screened proteins released from CNS myelin by phospholipase treatment for their ability to inhibit neurite outgrowth. The only inhibitory protein identified was OMgp. A further study suggests that it can in vitro induce growth cone collapse. OMgp competes with Nogo-66 and myelin-associated glycoprotein(MAG) for Nogo-receptor binding. It seems likely that MAG, Nogo-66 and OMgp share the same active site on NgR as part of their overlapping binding sites and mediate their effects through the same pathways though they have no sequence homology .Comparison of the deduced amino acid sequences of OMgp in 14 mammalian species reveals that they are very similar. Analysis of the sequence for conserved functional domains reveals that the protein is composed of a cysteine-rich amino-terminal leucine-rich repeat (CR) flanking domain, followed by a leucine-rich repeat domains (LRR) and a serine/threonine - rich region (S/T). The polypeptides are most similar in the leucin-rich domain , with more than 86% identity in the 14 species .The region corresponds to the LRR domain from amino acid 57-228 of the pre-protein, and 32 of the 34 leucines of this domain are conserved in all species. Given the high conservation, it is reasonable to speculate that this region is important for OMgp function.To better understand the structure-function relationship for the OMgp, a mouse OMgp cDNA was cloned and the distinct domain of OMgp was subcloned into pGEX-4T vector expressioin as GST-fusion protein. The NgR-expressing CHO and hippocampal neurons were in vitro cultured on these GST-fusion proteins. Results show that the protein fragment consisting of the S/T domain and C-terminal of OMgp could not bind with NgR-expressing CHO cells. The fragments representing LRR could bound with NgR-expressing CHO cells and inhibit the neurite outgrowth of primary neurons. It could also pull down NgR from mouse brain. The results suggest that the LRR domain of OMgp is required and sufficient for binding of NgR and neurite outgrowth inhibition. The fragment containing part of LRR can also function as the full length OMgp protein , though less effective.To identify the most essential domain of OMgp in interacting with NgR, distinct OMgp leucine-rich repeats were deleted by PCR based site-directed mutagenesis. Themutant OMgp LRR were expressed as GST fusion proteins, which were then used to culture with NgR-expressing CHO cells and hippocampal neurons. Results show that deletion of OMgp LRR fragments deleted amino acid residues 25-56,57-133,134-180 did not interrupt its effects in binding to NgR-expressing CHO cells and inhibiting hippocampal neuron growth. The fragment with deletion of amino acid residues 181-228 could only bind with NgR-expressing CHO cells and lost the inhibitory effect on neurite outgrowth. This suggest that the OMgp amino acid 181-228 is the predominant domain in neurite outgrowth inhibition and the OMgp LRR fragment without this domain may be a potent reagent for encouraging regeneration in the adult CNS following injury.Because it is now unavailable to get the crystal structure of OMgp , homologous modeling was did . Results show that OMgp LRR has a very similar structure to YopM-A. This may help to understand the structure-function relationship of OMgp.