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Inhibitory activity of myelin-associated glycoprotein on sensory neurons is largely independent of NgR1 and NgR2 and resides within Ig-Like domains 4 and 5.

Wörter V, Schweigreiter R, Kinzel B, Mueller M, Barske C, Böck G, Frentzel S, Bandtlow CE - PLoS ONE (2009)

Bottom Line: Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth.Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG.Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAG's ability to bind to sialic acid did not interfere with its inhibitory activity.

View Article: PubMed Central - PubMed

Affiliation: Innsbruck Medical University, Biocenter, Division of Neurobiochemistry, Innsbruck, Austria.

ABSTRACT
Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-like lectin (Siglec) which has been characterized as potent myelin-derived inhibitor of neurite outgrowth. Two members of the Nogo-receptor (NgR) family, NgR1 and NgR2, have been identified as neuronal binding proteins of MAG. In addition, gangliosides have been proposed to bind to and confer the inhibitory activity of MAG on neurons. In this study, we investigated the individual contribution of NgRs and gangliosides to MAG-mediated inhibition of sensory neurons derived from dorsal root ganglia (DRG) of ngr1, ngr2 or ngr1/ngr2 deletion mutants. We found no disinhibition of neurite growth in the absence of either NgR1 or NgR2. Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth. If treated with Vibrio cholerae neuraminidase (VCN), inhibition by MAG is further attenuated but still not aned. Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG. Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAG's ability to bind to sialic acid did not interfere with its inhibitory activity. These findings provide new insights into the inhibitory function of MAG and suggest similarities but also major differences in MAG inhibition between sensory and central nervous system (CNS) neurons.

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Inhibition of neurite outgrowth by a MAG mutant lacking the sialic acid binding site.(A) The name MAG R118A refers to a point-mutation within the sialic-acid binding domain of MAG (asterisk), that abolishes MAG binding to specific gangliosides. (B) Western Blot analysis of a CHO clone stably expressing MAG R118A. Ten micrograms of total protein lysate were loaded for each lane. Goat anti-MAG antibody was used to detect MAG; to reveal equal protein load, blots were incubated with anti-flotillin antibody. (C) Cell surface expression of full-length MAG and MAG R118A proteins by transfected CHO cells after FACS and clonal selection as revealed by live-staining with anti-MAG antibody. Scale bar 20 µm. (D) Analysis of neurite length of wildtype and ngr1/ngr2 double knock-out DRG neurons grown on CHO cell monolayers expressing full-length MAG or MAG R118A. Parental CHO cells served as the control substrate. Graph shows % neurite length on the different substrates±SEM. Three separate experiments were examined for each experimental condition, with 50 to 100 neurons measured for each substrate. *** represents P≤0.001 compared to CHO-K1, one-way analysis of variance with post-hoc Tukey test.
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pone-0005218-g006: Inhibition of neurite outgrowth by a MAG mutant lacking the sialic acid binding site.(A) The name MAG R118A refers to a point-mutation within the sialic-acid binding domain of MAG (asterisk), that abolishes MAG binding to specific gangliosides. (B) Western Blot analysis of a CHO clone stably expressing MAG R118A. Ten micrograms of total protein lysate were loaded for each lane. Goat anti-MAG antibody was used to detect MAG; to reveal equal protein load, blots were incubated with anti-flotillin antibody. (C) Cell surface expression of full-length MAG and MAG R118A proteins by transfected CHO cells after FACS and clonal selection as revealed by live-staining with anti-MAG antibody. Scale bar 20 µm. (D) Analysis of neurite length of wildtype and ngr1/ngr2 double knock-out DRG neurons grown on CHO cell monolayers expressing full-length MAG or MAG R118A. Parental CHO cells served as the control substrate. Graph shows % neurite length on the different substrates±SEM. Three separate experiments were examined for each experimental condition, with 50 to 100 neurons measured for each substrate. *** represents P≤0.001 compared to CHO-K1, one-way analysis of variance with post-hoc Tukey test.

Mentions: A characteristic feature of MAG is its sugar binding ability and it was investigated early on whether the lectin activity of MAG is related to its inhibitory effect on neurons. Previous studies showed that Arg118 in Ig domain 1 is crucial for sialic acid binding of MAG [3], but that the sialic acid binding ability of MAG is not necessary for inhibition [3]. The latter experiments were carried out with CGN and we wondered whether sialic acid binding of MAG is of any significance for conferring inhibitory activity on sensory neurons. By site-directed mutagenesis we constructed a MAG mutant that had Arg118 replaced with Ala, termed MAG R118A (Fig. 6A). A CHO clone that stably expressed MAG R118A on the cell surface (Fig. 6B, C) was used as a substrate for sensory neurons. No difference in the neurons' responsiveness to the MAG mutant in comparison to wildtype MAG was observed neither with wildtype nor ngr1/ngr2 double knock-out neurons (Fig. 6D), confirming that the sialic acid binding ability of MAG is not necessary for its inhibitory activity on neurons.


Inhibitory activity of myelin-associated glycoprotein on sensory neurons is largely independent of NgR1 and NgR2 and resides within Ig-Like domains 4 and 5.

Wörter V, Schweigreiter R, Kinzel B, Mueller M, Barske C, Böck G, Frentzel S, Bandtlow CE - PLoS ONE (2009)

Inhibition of neurite outgrowth by a MAG mutant lacking the sialic acid binding site.(A) The name MAG R118A refers to a point-mutation within the sialic-acid binding domain of MAG (asterisk), that abolishes MAG binding to specific gangliosides. (B) Western Blot analysis of a CHO clone stably expressing MAG R118A. Ten micrograms of total protein lysate were loaded for each lane. Goat anti-MAG antibody was used to detect MAG; to reveal equal protein load, blots were incubated with anti-flotillin antibody. (C) Cell surface expression of full-length MAG and MAG R118A proteins by transfected CHO cells after FACS and clonal selection as revealed by live-staining with anti-MAG antibody. Scale bar 20 µm. (D) Analysis of neurite length of wildtype and ngr1/ngr2 double knock-out DRG neurons grown on CHO cell monolayers expressing full-length MAG or MAG R118A. Parental CHO cells served as the control substrate. Graph shows % neurite length on the different substrates±SEM. Three separate experiments were examined for each experimental condition, with 50 to 100 neurons measured for each substrate. *** represents P≤0.001 compared to CHO-K1, one-way analysis of variance with post-hoc Tukey test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666269&req=5

pone-0005218-g006: Inhibition of neurite outgrowth by a MAG mutant lacking the sialic acid binding site.(A) The name MAG R118A refers to a point-mutation within the sialic-acid binding domain of MAG (asterisk), that abolishes MAG binding to specific gangliosides. (B) Western Blot analysis of a CHO clone stably expressing MAG R118A. Ten micrograms of total protein lysate were loaded for each lane. Goat anti-MAG antibody was used to detect MAG; to reveal equal protein load, blots were incubated with anti-flotillin antibody. (C) Cell surface expression of full-length MAG and MAG R118A proteins by transfected CHO cells after FACS and clonal selection as revealed by live-staining with anti-MAG antibody. Scale bar 20 µm. (D) Analysis of neurite length of wildtype and ngr1/ngr2 double knock-out DRG neurons grown on CHO cell monolayers expressing full-length MAG or MAG R118A. Parental CHO cells served as the control substrate. Graph shows % neurite length on the different substrates±SEM. Three separate experiments were examined for each experimental condition, with 50 to 100 neurons measured for each substrate. *** represents P≤0.001 compared to CHO-K1, one-way analysis of variance with post-hoc Tukey test.
Mentions: A characteristic feature of MAG is its sugar binding ability and it was investigated early on whether the lectin activity of MAG is related to its inhibitory effect on neurons. Previous studies showed that Arg118 in Ig domain 1 is crucial for sialic acid binding of MAG [3], but that the sialic acid binding ability of MAG is not necessary for inhibition [3]. The latter experiments were carried out with CGN and we wondered whether sialic acid binding of MAG is of any significance for conferring inhibitory activity on sensory neurons. By site-directed mutagenesis we constructed a MAG mutant that had Arg118 replaced with Ala, termed MAG R118A (Fig. 6A). A CHO clone that stably expressed MAG R118A on the cell surface (Fig. 6B, C) was used as a substrate for sensory neurons. No difference in the neurons' responsiveness to the MAG mutant in comparison to wildtype MAG was observed neither with wildtype nor ngr1/ngr2 double knock-out neurons (Fig. 6D), confirming that the sialic acid binding ability of MAG is not necessary for its inhibitory activity on neurons.

Bottom Line: Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth.Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG.Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAG's ability to bind to sialic acid did not interfere with its inhibitory activity.

View Article: PubMed Central - PubMed

Affiliation: Innsbruck Medical University, Biocenter, Division of Neurobiochemistry, Innsbruck, Austria.

ABSTRACT
Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-like lectin (Siglec) which has been characterized as potent myelin-derived inhibitor of neurite outgrowth. Two members of the Nogo-receptor (NgR) family, NgR1 and NgR2, have been identified as neuronal binding proteins of MAG. In addition, gangliosides have been proposed to bind to and confer the inhibitory activity of MAG on neurons. In this study, we investigated the individual contribution of NgRs and gangliosides to MAG-mediated inhibition of sensory neurons derived from dorsal root ganglia (DRG) of ngr1, ngr2 or ngr1/ngr2 deletion mutants. We found no disinhibition of neurite growth in the absence of either NgR1 or NgR2. Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth. If treated with Vibrio cholerae neuraminidase (VCN), inhibition by MAG is further attenuated but still not aned. Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG. Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAG's ability to bind to sialic acid did not interfere with its inhibitory activity. These findings provide new insights into the inhibitory function of MAG and suggest similarities but also major differences in MAG inhibition between sensory and central nervous system (CNS) neurons.

Show MeSH
Related in: MedlinePlus