Characterization of the N-terminal region of Crtac1B/LOTUS, an endogenous Nogo receptor-1 antagonist, required for secretion and soluble function

Abstract

Lateral olfactory tract usher substance (LOTUS), also known as cartilage acidic protein-1B (Crtac1B), is a potent endogenous antagonist of Nogo receptor type-1 (NgR1). LOTUS exists in both membrane-bound (m-LOTUS) and soluble (s-LOTUS) forms. m-LOTUS inhibits NgR1 by binding to its C-terminal domain (UA-EC domain), thereby blocking ligand interactions, whereas s-LOTUS disrupts NgR1 signaling by interfering with its interaction with the p75 co-receptor. However, the molecular characteristics of s-LOTUS, including the functional domains required for secretion and NgR1 binding, remain unclear.

In this study, we identified the N-terminal 35Met-532Pro region as essential for s-LOTUS secretion. While N-type glycosylation patterns remained unchanged, differences in intracellular localization with the Golgi apparatus were observed between secretory and non-secretory forms. The non-secretory form exhibited higher aggregation and ubiquitination levels. Functionally, the soluble 35Met-532Pro fragment bound to both NgR1 and p75, disrupting the NgR1-p75 complex. In contrast, the UA-EC domain, corresponding to the functional domain of m-LOTUS, bound only to NgR1 and did not interfere with p75 interaction.

A growth cone collapse assay using cultured olfactory bulb neurons from lotus-deficient embryonic mice demonstrated that exogenous 35Met-532Pro, but not UA-EC, inhibited Nogo66-induced growth cone collapse. These findings indicate that the N-terminal 35Met-532Pro region of s-LOTUS functions as its active domain, antagonizing NgR1 signaling through dual binding to NgR1 and p75. In contrast, the UA-EC domain lacks this antagonistic property, highlighting the distinct functional domains and mechanisms of action between s-LOTUS and m-LOTUS.