Calcium-binding protein, spermatid-specific 1 is expressed in human salivary glands and contains an anti-inflammatory motif

Abstract

Salivary glands are involved in the production and exocrine and endocrine secretion of biologically active proteins, polypeptides, and hormones involved in growth and differentiation, homeostasis, and digestion. We have previously studied the prohormone submandibular rat 1 (SMR1), product of the Vcsa1 gene, which is highly expressed in the testes and salivary glands of rats, and can be cleaved to produce polypeptides with analgesic, erectile function, and anti-inflammatory activities. Humans lack the Vcsa1 gene, but homologous sequences and functions for analgesia and erectile function exist in the human genes Prol1, SMR3a, and SMR3b located on the human chromosomal region close to where Vcsa1 lies in the rat. Here we show the human protein calcium-binding protein spermatid-specific 1 (CABS1) contains a similar sequence to the anti-inflammatory sequence in rat SMR1, thus CABS1 may be another human gene with homologous function to Vcsa1. Using Western blot and PCR, we discovered that the human protein CABS1, previously thought to only be expressed in the testes, is also expressed in the salivary glands and lung, in a tissue-specific manner. Peptides derived from CABS1 were tested in an in vivo mouse model of lipopolysaccharide (LPS)-induced neutrophilia and an ex vivo rat model of antigen-induced intestinal anaphylaxis and significantly reduced both neutrophil accumulation in bronchoalveolar lavage fluid and antigen-induced ileal contractions, respectively. Thus human CABS1 has a peptide motif homologous to the anti-inflammatory peptide sequence of rat SMR1. Whether this similarity of CABS1 extends to the neuroendocrine regulation of the anti-inflammatory activity seen for SMR1 remains to be determined.

Keywords: SMR1; Vcsa1; inflammation; neutrophilia; saliva.

Fig. 1.

CABS1, SMR3a, SMR3b, and PROL1 contain functional motifs with biological activity homologous to rat Vcsa1. Shown are the genes in this cluster on chromosome 4 of Homo sapiens and chromosome 14 of Rattus norvegicus. This region is well conserved in mammals and the known tissue expression for the human genes is indicated above. Rat and human homologues are joined with dotted lines. Solid lines indicate functional homologues of Vcsa1 that have acquired sequences with similar biological functions. From Morris et al. (23).

Fig. 2.

CABS1 mRNA is expressed in human salivary gland and testes. mRNA was isolated from human submandibular glands (SMG, lanes 1–5), or commercially purchased total RNA (SMG, lane 6; parotid, lane 7; testes, lane 8). cDNA was made and PCR was done using CABS1-specific primers. RNA (0.25 μg) was used in the testes reaction and 2 μg in all others. PCR products were run on a 1.5% agarose gel and visualized using ethidium bromide. Male samples are in lanes 1, 3, 4, 6, 7, and 8 and female samples are in lanes 2 and 5.

Fig. 3.

CABS1 is expressed in human submandibular glands, testes, and lung. Protein was analyzed from five human SMG, testes, lung, and spleen (A) and SMG, control and overexpression (OE) lysates, and purified CABS1 (B). Lysate (25 μg) or purified CABS1 (1 μg) was run on 12% polyacrylamide gels. The membranes were blotted with CABS1 antibody H2. In A, male SMG samples are in lanes 1, 3, and 4 and female samples are in lanes 2 and 5. Numbers on left are the relative molecular mass (M_r) of each band in kilodaltons (kDa). Red bands are β-actin and green bands are CABS1.

Fig. 4.

CABS1-derived peptides have anti-anaphylactic activity in an intestinal antigen challenge model. The terminal ileum was excised from OA-sensitized rats, and segments were mounted in an organ bath connected to a force displacement transducer. Ileal segments were incubated with two doses of peptides (10⁻⁶ M and 10⁻⁷ M) for 10 min, followed by antigen challenge with 1 mg OA, and then ileal contractions were measured. Peak contractile response of each ileal segment was measured by adding 10⁻⁵ M carbachol, and all results are expressed as the OA-to-Carbachol contractile ratio. Peptides tested were fdG (Phe, Asp, Gly) (A), feG (Phe, Glu, Gly) (B), FELL (C), TDIFELLK (D), and TDIFELL (E); n = 13–29 animals in A and B and n = 12–23 animals in C–E. Significance is represented as *P < 0.05 and **P < 0.01.

Fig. 5.

CABS1-derived peptides have anti-inflammatory activity. Mice were pretreated with 5 mg/kg peptide by gavage followed 1 h later by 500 μg/kg lipopolysaccharide (LPS) instilled intranasally. Twenty-four hours later bronchoalveolar lavage (BAL) was performed and total white blood cells (WBC, A), neutrophils (B), and macrophages (C) in the BAL fluid were counted. To compensate for interexperimental variability, results are normalized to our LPS-positive control group for each experiment; n = 6–10 animals in each group. Significance is represented as *P < 0.05, **P < 0.01, and ***P < 0.001.