The carboxyl-terminal region of Crtac1B/LOTUS acts as a functional domain in endogenous antagonism to Nogo receptor-1

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Abstract

Myelin-derived axon growth inhibitors, such as Nogo, bind to Nogo receptor-1 (NgR1) and thereby limit the action of axonal regeneration after injury in the adult central nervous system. Recently, we have found that cartilage acidic protein-1B (Crtac1B)/lateral olfactory tract usher substance (LOTUS) binds to NgR1 and functions as an endogenous NgR1 antagonist. To examine the functional domain of LOTUS in the antagonism to NgR1, analysis using the deletion mutants of LOTUS was performed and revealed that the carboxyl-terminal region (UA/EC domain) of LOTUS bound to NgR1. The UA/EC fragment of LOTUS overexpressed together with NgR1 in COS7 cells abolished the binding of Nogo66 to NgR1. Overexpression of the UA/EC fragment in cultured chick dorsal root ganglion neurons suppressed Nogo66-induced growth cone collapse. These findings suggest that the UA/EC region is a functional domain of LOTUS serving for an antagonistic action to NgR1.

Highlights

► Two carboxyl-terminal regions of LOTUS bind to NgR1. ► The carboxyl-terminal regions of LOTUS block the binding of Nogo66 to NgR1. ► The carboxyl-terminal regions of LOTUS inhibit Nogo66-induced growth cone collapse.

Introduction

It has been widely accepted that neurons in the adult central nervous system (CNS) limit axonal regeneration after injury. This limitation can be attributed to inhibitory environmental factors such as axon growth inhibitors derived from myelin. Nogo proteins, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) are well known as myelin-derived axon growth inhibitors [1], [2]. Nogo receptor-1 (NgR1) is a glycosylphosphatidylinositide (GPI)-anchored protein and a common receptor for at least these three myelin components [1], [2]. Accumulating lines of evidence have shown that neutralization of these inhibitors’ function by antibodies [3], soluble NgR1 peptides [4], an NgR1 antagonist [5] or gene deletion [6], [7] improves axonal regeneration after injury.

Recently, we identified cartilage acidic protein-1B (Crtac1B)/lateral olfactory tract usher substance (LOTUS), which binds specifically to NgR1, and found that LOTUS functions in lateral olfactory tract development through an antagonism to NgR1 [8]. LOTUS is a 646 amino acid protein and consists of a signal peptide sequence, four phenylalanyl–glycyl and glycyl–alanyl–prolyl (FG-GAP) domains, a UnbV/ASPIC (UA) domain, an epidermal growth factor-like calcium binding (EC) domain and a transmembrane (TM) domain that is located at the carboxyl-terminus [8], [9], [10]. However, the region of LOTUS involved in the antagonistic action to NgR1 has not been determined. In this study, we identified the region of LOTUS that binds to NgR1 by using the deletion mutants of LOTUS, and we examined whether the domain exerts antagonism to NgR1. Our results demonstrate that two carboxyl-terminal domains of LOTUS bind to NgR1 and act as a functional domain in antagonism to NgR1.

Section snippets

Animals

Fertilized White Leghorn eggs were purchased from Yamagishi (Gumma, Japan) and incubated at 37 °C in a standard egg incubator. Dorsal root ganglions (DRGs) were dissected from chick embryos of indeterminate sex at embryonic day 13 (E13). Throughout the experimental procedures, all efforts were made to minimize the number of animals used and their suffering. The experimental procedures were approved by the institutional animal care and use ethical committee of the Yokohama City University School

Two carboxyl-terminal regions of LOTUS bind to NgR1

To examine the binding region of LOTUS to NgR1, we generated amino-terminal AP-fused deletion mutants of LOTUS and analyzed their binding properties for NgR1 expressed in COS7 cells (Fig. 1A). The proteins containing the TM region of LOTUS were also secreted into the culture medium for unknown reasons. The proteins were possibly severed near the TM region of LOTUS, because the molecular size of secreted proteins was almost equal to that of the membrane protein (data not shown). No binding of AP

Discussion

Previous studies show that axon regeneration in the injured adult CNS is promoted by negatively regulating NgR1 [3], [4], [5], [6], [7]. We have recently shown that LOTUS binds to NgR1 and functions as an endogenous antagonist to NgR1 [8]. We here determined that two carboxyl-terminal segments of LOTUS act as a functional region in antagonism to NgR1.

The UA domain or the EC domain alone contributes to the interaction between LOTUS and NgR1, whereas the combination of both domains (UA/EC) binds

Acknowledgments

This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sport, Science, and Technology of Japan (to K.T.) and by the Grants for Research and Development Project II (No. S2107) of Yokohama City University (to Y.G. and K.T.). We are grateful to Drs. Y. Sasaki and N. Yamashita at Yokohama City University for helpful discussions and Ms. H. Sekiguchi-Kaneko, M. Ogawara, T. Okada and M. Matsuura for technical assistance.

References (26)

  • P. Freund et al.

    Nogo-A-specific antibody treatment enhances sprouting and functional recovery after cervical lesion in adult primates

    Nat. Med.

    (2006)
  • S. Li et al.

    Blockade of Nogo-66, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein by soluble Nogo-66 receptor promotes axonal sprouting and recovery after spinal injury

    J. Neurosci.

    (2004)
  • S. Li et al.

    Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury

    J. Neurosci.

    (2003)
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