Chemical Synthesis of NICOL, A Coligand Secreted Protein Mediating Mammalian Male Reproductive Tract Trans-luminal Signaling

Abstract

The NELL2-interacting cofactor for lumicrine signaling (NICOL) is a small protein which forms a complex with the neural epidermal growth factor-like like 2 (NELL2) and engaged in sperm maturation in the epididymis. At present, the recombinant expression of NICOL is not efficient, which limits the precise understanding of the structure and function of NICOL. In this study, we first succeeded in the chemical synthesis of NICOL with NELL2 binding affinity. The synthesized NICOL possesses correct SS bond pairing, and its secondary structure is indistinguishable from that of the recombinant protein, indicating that the synthetic NICOL will be useful for structural and functional analyses of lumicrine signaling.

Keywords: NELL2; NICOL; chemical synthesis; lumicrine.

Figure 1

A sequence alignment of NICOL proteins of multiple species. UniProt entries of NICOL proteins are as follows: M.musculus (Q3UR78), H.sapiens (Q5BLP8), G.gallus (E1BRC3), A.carolinensis (H9GV37), X.tropicalis (A0A803J543), D.rerio (E7FEX9). Signal sequences are indicated in italics. Conserved Cys residues are highlighted with yellow. Potential O‐glycosylation sites are highlighted with cyan.

Figure 2

RPHPLC profile of the crude product 2 obtained by the SPPS and the ones 3 and 4 obtained after the oxidative folding reaction. Column: Cosmosil Protein R (4.6 x 250 mm), eluent: MeCN aq containing 0.1% TFA at the flow rate of 1.0 mL•min⁻¹.

Figure 3

SS bond determination procedure of peptide 3 a) and the RPHPLC profile of the reaction b). Column: Mightysil RP‐18 GP II (4.6 x 150 mm), eluent: MeCN aq containing 0.1% TFA at the flow rate of 1.0 mL•min⁻¹.

Figure 4

Synthetic procedure for NICOL 3 by the selective SS bond formation a) and the results of the synthesis b). The elution conditions are the same as those in Figure 3.

Figure 5

NELL2 pulldown assay using recombinant and synthetic NICOL. Immunoblot analysis of NELL2 pulled down with recombinant NICOL 1, two synthetic NICOL 3 and 4, and BSA (negative control). Input NELL2 protein was also included as a reference. The uncropped image is shown in Figure S15.

Figure 6

CD spectrum of NICOLs measured in MES buffer (pH 6.0).