Neurexophilins form a conserved family of neuropeptide-like glycoproteins

Abstract

Neurexophilin was discovered as a neuronal glycoprotein that is copurified with neurexin Ialpha during affinity chromatography on immobilized alpha-latrotoxin (Petrenko et al., 1996). We have now investigated how neurexophilin interacts with neurexins, whether it is post-translationally processed by site-specific cleavage similar to neuropeptides, and whether related neuropeptide-like proteins are expressed in brain. Our data show that mammalian brains contain four genes for neurexophilins the products of which share a common structure composed of five domains: an N-terminal signal peptide, a variable N-terminal domain, a highly conserved central domain that is N-glycosylated, a short linker region, and a conserved C-terminal domain that is cysteine-rich. When expressed in pheochromocytoma (PC12) cells with a replication-deficient adenovirus, neurexophilin 1 was rapidly N-glycosylated and then slowly processed to a smaller mature form, probably by endoproteolytic cleavage. Similar expression experiments in other neuron-like cells and in fibroblastic cells revealed that N-glycosylation of neurexophilin 1 occurred in all cell types tested, whereas proteolytic processing was observed only in neuron-like cells. Finally, only recombinant neurexin Ialpha and IIIalpha but not neurexin Ibeta interacted with neurexophilin 1 and were preferentially bound to the processed mature form of neurexophilin. Together our data demonstrate that neurexophilins form a family of related glycoproteins that are proteolytically processed after synthesis and bind to alpha-neurexins. The structure and characteristics of neurexophilins indicate that they function as neuropeptides that may signal via alpha-neurexins.

Fig. 1.

Primary structure of neurexophilins from rat (R), mouse (M), bovine (B), and human (H). The amino acid sequences of the four neurexophilins (NPH1–NPH4) are aligned for maximal homology, withhyphens indicating gaps. Sequences are identified on theleft and numbered on the right. Residues that are identical in all sequences are shown on a redbackground, and residues identical in at least two isoforms are shown on a blue background. The four conserved N-glycosylation sites in neurexophilins are identified by arrows, and the six cysteines present in all neurexophilins with identical spacing are marked by asterisks above the sequences. The putative signal sequence is shown in italics. The linker sequence in neurexophilin 4 between the N-glycosylation domain and the cysteine-rich domain contains seven imperfect GGxL repeats (residues 201–234). The human neurexophilin 1, 3, and 4 sequences were obtained from incomplete EST clones and are only partial. No rat neurexophilin 2 cDNA could be identified, leading to the absence of a rat neurexophilin 2 sequence; the mouse neurexophilin 2 sequence was deduced from the genomic sequence (Petrenko et al., 1996).

Fig. 2.

Domain model of neurexophilins based on their primary structures. Five domains are proposed (identified by roman numerals): I, an N-terminal signal peptide; II, a variable N-terminal region that may be cleaved off during proteolytic processing; III, a highly conserved domain containing three N-glycosylation sites; IV, a linker sequence that varies in size and composition between different neurexophilins; and V, a conserved C-terminal domain with six cysteine residues that are identically spaced in all neurexophilins. The putative site of proteolytic cleavage is indicated by an arrow above the diagram, positions of N-glycosylation sequences are marked by branched lines, and the conserved cysteines are identified by letters C. The size range of the different domains in the neurexophilins and the sequence identities and homologies between neurexophilins in the domains are shownbelow the diagram. n.a., Not applicable.

Fig. 3.

Tissue-specific expression of neurexophilins in rats, mice, and humans. RNA blots are hybridized with neurexophilin 1 (Nph 1; A, E,I), neurexophilin 2 (Nph 2;B, F, J), neurexophilin 3 (Nph 3; C,G, K), and neurexophilin 4 (Nph 4; D, H,L) probes. RNA blots from mouse (A–D), rat (E–H), and human (I–L) tissues contained total RNA from heart (H; lane 1), brain (B; lane 2), spleen (S;lane 3), lung (Lu; lane 4), liver (Li; lane 5), skeletal muscle (Sk; lane 6), kidney (K; lane 7), and testis (T; lane 8). A single multitissue RNA blot (obtained from Clontech) from each species was consecutively hybridized with probes for each neurexophilin. Rat probes were used for all hybridizations except for the blots for human neurexophilins 3 and 4 (K and L) that were hybridized with human probes. Arrowheads for D andH mark the position of the 28S RNA that cross-hybridizes with the glycine- and cysteine-rich rat neurexophilin 4 probe. Positions of molecular size markers are indicated on theleft.

Fig. 4.

Time-dependent proteolytic processing of neurexophilin 1. PC12 cells were infected with recombinant adenovirus encoding full-length neurexophilin 1 (AdNph1), and neurexophilin expression was analyzed as a function of time by SDS-PAGE and immunoblotting. To control for protein loads, we then reprobed the same blot for synaptotagmin I. Without adenovirus infection (lane 1), only synaptotagmin I but no neurexophilin can be detected. Full-length neurexophilin corresponding to the N-glycosylated form (Nph 1[unprocessed]) is detected within 24 hr of infection (lanes 4–6), whereas the shorter, processed form of neurexophilin (Nph 1 [processed]) appears later (lanes 7–9) and becomes the dominant form only after 3 d (lanes 10–12). No processing is observed with neurexophilin expressed in COS cells (lane 13).

Fig. 5.

N-glycosylation of neurexophilin 1 expressed in PC12 cells. PC12 cells infected with recombinant adenovirus encoding neurexophilin 1 (AdNph1) were lysed 2 d after infection, and lysates were treated with 1000 or 5000 units of endoglycosidase F (PNGaseF) or with control buffer. Samples were analyzed by SDS-PAGE and immunoblotting, demonstrating a similar shift of the unprocessed and processed forms of neurexophilin by endoglycosidase F treatment but not by control treatment. Numbers on the left indicate positions of molecular weight standards.

Fig. 6.

Proteolytic processing of neurexophilin 1 in different cell types. Cell lines of neuronal [hNT (A) and SH-SY5Y (B) cells] and non-neuronal [STO (C) and COS (D) cells] origin were infected with different concentrations (pfu, plaque-forming units) of recombinant adenovirus expressing neurexophilin 1. In addition, neurexophilin 1 was also expressed in the non-neuronal cells by transfection. The size of recombinant neurexophilin 1 produced in the various cell types was analyzed by SDS-PAGE and immunoblotting and compared with that produced in PC12 cells infected with the neurexophilin 1 adenovirus. Numbers on the left of eachpanel indicate positions of molecular weight markers;arrows on the right mark the migration of unprocessed (top) and processed (bottom) neurexophilin.

Fig. 7.

Binding of recombinant neurexophilin 1 to neurexin Iα. PC12 cells infected with recombinant adenovirus expressing neurexophilin 1 were incubated for 24 hr, the earliest time at which mature processed neurexophilin is detected (lane 1). Immobilized IgG fusion proteins of either neurexin Iβ (NxIβ-1; lane 2), a short irrelevant sequence (control; lane 3), or rat and bovine neurexin Iα (NxIα-1; lanes 4,5) were incubated with lysates from infected PC12 cells, and bound proteins were analyzed by SDS-PAGE and immunoblotting for neurexophilin. Numbers on the left indicate positions of molecular weight markers. Note that both processed and unprocessed neurexophilin specifically binds only to neurexin Iα but that there is a relative enrichment of processed neurexophilin in the bound fraction.