Characterization of common salivary protein 1, a product of rat submandibular, sublingual, and parotid glands.

L R Girard,A M Castle,A R Hand,J D Castle,L Mirels

doi:10.1016/s0021-9258(19)74353-1

L R Girard, A M Castle + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(19)74353-1

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Abstract

We have isolated and characterized cDNA clones derived from a developmentally regulated neonatal rat submandibular gland salivary protein gene called "common salivary protein 1" (CSP1). Identical clones were also identified in cDNA libraries from adult male parotid, submandibular, and sublingual glands. CSP1 transcripts are at least 10-fold more abundant in the sublingual gland than in the submandibular or parotid glands. In situ hybridization and immunocytochemical localization demonstrated the presence of CSP1 transcripts and proteins in sublingual gland serous demilune cells, parotid and submandibular gland intercalated duct cells, and in the type III (proacinar) cells of the neonatal submandibular gland. This cell-type distribution is similar to that described by Ball and colleagues (Ball, W. D., Hand, A. R., and Johnson, A. O. (1988) Dev. Biol. 125, 265-279) for the developmentally regulated submandibular gland B1-immunoreactive proteins. Immunoblotting of salivary secretion identified proteins of M(r) 20,000 in sublingual, 16,000 in submandibular and 22,000 and 16,000 in parotid gland. The M(r) 20,000 sublingual and 22,000 parotid proteins represent N-glycosylated forms of a M(r) 16,000 apoprotein, suggesting that these salivary proteins arise by post-translational modification of a common precursor.

Highlights

Salivary proteins produced by the acinar cells of the major salivary glands are secreted into networks of coalescing ducts that lead to the oral cavity
CSPl Is DevelopmentallyRegulated in the Neonatal Rat Submandibular Gland-The rat salivarytranscripts identified in this report encode a protein designated common salivary protein 1or CSP1,based on its expression in cells of all major salivary glands
A CSPl cDNA wasfirst identified in a library screen designed to identify developmentally regulated products of the neonatal submandibular gland

Summary

EXPERIMENTAL PROCEDURES

Annealed a t 42 "C for 8 h, and reverse transcription was performed using M-MuLV reverse transcriptase accordingto the manufacturer's. SDS (for first strand cDNA) or 0.1 X SSC, 0.1% SDS for cDNA probes and used to expose Kodak XAR-5 film for 20 h at -80 'C with an intensifying screen Plaques identified in this manner were isolated. Gland (for immunocytochemistry) as described [37] and incubated in DNA Sequence Analysis-CSPI cDNA clones were sequenced by the presence of glutathione-S-transferase-conjugatedSepharose CL-. Tissue sections were pretreated as described [48], blocked with 10%nonimmune goat serum in PBSfor 30 min at room temperature, and incubated with anti-glutathioneS-transferaseCSPl in PBS with 1.5% nonimmune goat serum a t 4 "C overnight. The slides were washed and prehybridized in 40% formamide, 5 X SSC, 1 X Denhardt's, 500 pg/ml salmon sperm DNA, 100 pg/ml yeast tRNA, 10% dextransulfate, 0.5% nucleic acid blocking reagent (Boehringer Mannheim) at room temperature for 2 h. After thorough rinsing with PBS and distilled water, the sections were stained with uranyl acetate and lead citrate and examined in a Philips CMlO transmission electron microscope operated at 60 kV

RESULTS

Asn Val Asn Glv ThrCys Thr Glu * * *

Characterizationof C S P l

Endo H

DISCUSSION