Immunological comparison of proline-rich proteins from human and primate parotid secretion.

Antisera raised in response to proline-rich proteins purified from parotid secretions of man and the primate Macaca fascicularis were employed to investigate the interrelationships of these proteins by immunodiffusion, immunoelectrophoresis and the combined use of disc gel acrylamide electrophoresis with radial immunodiffusion. The major human proline-rich proteins, PRP I, PRP II, PRP III and PRP IV as well as several minor proline-rich proteins cross-react with antiserum to PRP I or PRP III. Similarly primate parotid saliva contains several components cross-reacting with antiserum directed against a purified primate proline-rich protein, MPRP. Antiserum to PRP I or PRP III cross-reacted with MPRP and primate parotid saliva protein, whereas antiserum to MPRP cross-reacted only with human parotid saliva protein and not with the isolated human proline-rich proteins. The immunological relationships of these salivary proline-rich proteins within and between species suggest their origin from a common precursor molecule.

Sequence and expression of the MnP4 gene encoding basic proline-rich protein in macaque salivary glands.

We report here the macaque MnP4 cDNA and genomic sequences which encode a basic proline-rich protein (PRP), which is synthesized in macaque parotid gland and submandibular gland. The locations of intron positions and the prototype of the tandem 20-amino-acid repeat motif with the sequence, PPPPGKPQGPPQQGGNKPQG, in MnP4, were compared to those in related genes encoding PRP and glutamic/glutamine-rich proteins (GRP) in humans and rodents. Exceedingly high homology of the first exon and 40-bp region immediately upstream of exon I is observed with other PRP genes of all species studied. In order to identify the regulatory elements involved in control of MnP4 gene expression, a rat submandibular gland-derived cell line (RSMT-A5) was transfected with MnP4-cat constructs that contained the promoter and 5'-flanking regions of the macaque MnP4 gene fused to the bacterial cat gene. Deletion analysis revealed that putative positive and negative regulatory elements reside between nucleotides (nt) -107 and +5, and nt -586 and -108, respectively. As part of this study, the promoter of the macaque MnP4 gene appears to be salivary gland specific. This salivary gland-specific gene expression attests to the complexity of transcriptional regulation in eukaryotes.

Beta-adrenergic regulation of c-fos gene expression in an epithelial cell line.

Stimulation of beta-adrenoreceptors in the RSMT-A5 epithelial cell line is accompanied by an early and transient increase in the expression of the proto-oncogene c-fos. Maximal induction was at 30 min, returning to basal levels after 2 h. Similar results were obtained when cells were incubated with 8-bromo-cAMP. The induction of c-fos is specific since the expression of p53, a transformation-related gene, is not modulated by isoproterenol or 8-bromo-cAMP. The increase in c-fos gene expression is not associated with proliferative activity in these epithelial cells.

N-linked protein glycosylation in the rat parotid gland during aging.

N-Linked protein glycosylation was examined in vitro in dispersed rat parotid acinar cells from young adult (3-6 months) and aged (22-24 months) rats. A small decrease in general protein production was observed with cells from aged animals (approximately 20% lower incorporation of [14C]leucine into 10% CCl3 COOH insoluble protein during continuous pulse labeling). Incorporation of [3H]mannose into N-linked glycoproteins by aged cells was further reduced (approximately 35%). Similarly microsomal membranes from parotid glands of aged animals showed approximately 50% reduction in the synthesis of mannosylphosphoryl dolichol, a key intermediate in the dolichol pathway of protein N-glycosylation. Man-P-Dol synthase, the microsomal enzyme responsible for production of this saccharide-lipid, displayed no change in apparent Km for GDP-mannose when preparations from aged animals were utilized, but did show approximately 50% reduction in Vmax. Following beta-adrenoreceptor activation, cells from both young adult and aged glands showed increased N-linked protein glycosylation almost to the same extent (approximately 2-fold). The data suggested that in aged rat parotid cells there is a basal reduction of activity in the pathway responsible for asparagine-linked protein glycosylation, but that following exocytotic stimuli this pathway responds in a manner comparable to cells from young adult glands.

Ultrastructural localization of salivary acidic proline-rich proteins from Macaca fascicularis.

Using an indirect immunoferritin method, the subcellular distribution of acidic proline-rich proteins (MPRP) in the acinar cells of the macaque parotid and submandibular glands was defined. MPRP was found in the "spherule" substructure of the secretory granules as well as in specific Golgi transfer vesicles and in vesicles budding from the Golgi apparatus. The data suggest that the acidic proline-rich proteins of the primate Macaca fascicularis are synthesized and packaged by conventional exocrine mechanisms. In addition, these proteins appear to be packaged and transferred to the secretory granules of parotid and submandibular gland acinar cells as discrete aggregates that remain as a separate "spherule" area in the secretory granules.

Effects of ionizing radiation and beta-adrenergic stimulation on the expression of early response genes in rat parotid glands.

There is little known about the regulation of gene expression in rat parotid glands after exposure to ionizing radiation. The present studies investigate the effects of in vivo ionizing radiation, with subsequent stimulation of beta-adrenergic receptors by isoproterenol, on parotid gland function and on the expression of the early response genes, c-fos, c-jun, and jun B. Ionizing radiation diminished parotid gland weight and saliva output. Treatment of irradiated rats with isoproterenol increased the gland weight to levels similar to those in nonirradiated rats. However, such treatment had no effect on saliva output as indicated by measurements of parotid salivary flow rate. Irradiation alone increased the expression of c-fos, c-jun, and jun B. The combination of irradiation and isoproterenol had an additional effect on the levels of c-fos and jun B mRNAs and proteins particularly at earlier experimental times (1 to 8 h). Isoproterenol alone induced high levels of c-fos and jun B mRNA but not of c-jun mRNA. However, c-jun mRNA was induced markedly by radiation and 8 h of isoproterenol treatment, indicating a combined effect on c-jun gene expression. These observations suggest that the expression of the proto-oncogenes c-fos, c-jun, and jun B is probably regulated through differential signal transduction pathways which may be activated by these external stimuli and may be associated with functional changes induced in the rat parotid gland by ionizing radiation and by ionizing radiation and isoproterenol.

Immunochemical identification and determination of proline-rich proteins in salivary secretions, enamel pellicle, and glandular tissue specimens.

Acidic proline-rich proteins were localized histologically by means of the indirect immunoperoxidase technique in the acinar cells of parotid and submandibular gland sections of both man and Macaca fascicularis. These proteins were also identified as constituents of the long term in vivo-formed acquired pellicle on human tooth surfaces employing the same immunoperoxidase technique. Immunological quantitation of proline-rich proteins in whole and parotid saliva of 42 subjects indicates the presence of high concentrations in parotid secretions varying between 19-80 mg%, while their levels in whole saliva are significantly lower, ranging between 0-18 mg%. The concentrations of these proteins in whole saliva exhibited a negative correlation with plaque accumulation (r = -0.464) and gingival index scores (r = -0.576).

Regulatory aspects of N-linked glycoproteins.

Activation of beta-adrenoreceptors in rat parotid acinar cells leads to copious exocrine protein secretion. Additionally, beta-adrenergic stimulation dramatically increases specific secretory protein synthesis and enhances N-linked glycosylation of secretory glycoproteins. Recently, efforts have been directed toward understanding the mechanisms underlying these biosynthetic events. We have been particularly interested in the receptor-mediated regulation of glycosylation. In this report, we evaluate available mechanistic information from the rat parotid gland and present initial data examining the ability of various regulatory agents to modulate N-linked glycosylation in enzymatically-dispersed cell aggregates from surgical specimens of human parotid glands. We conclude that glycosylation of human parotid N-linked glycoproteins may be regulated by extracellular signaling similar to that operative in the rat parotid gland.

Altered bacterial aggregation and adherence associated with changes in rat parotid-gland salivary proteins induced in vivo by beta-adrenergic stimulation.

Reduced adherence and aggregation were associated with protein alterations in parotid saliva after chronic treatment with the beta-adrenergic agonist isoproterenol. In contrast, saliva from animals treated with the beta-antagonist, propranolol, did not cause such changes; the protein composition of this saliva was similar to that of controls. SDS-polyacrylamide gel electrophoresis of protein in saliva samples before and after they were mixed with 10 mg of spheroidal hydroxyapatite beads (HA), as well as protein adsorbed and recovered from the HA, showed that an acidic, proline-rich protein with a molecular weight of approx. 40,000 was the predominant protein adsorbed. This protein was significantly diminished in saliva from isoproterenol-treated rats. Proteins with molecular weights between 44,000 and 48,000 and unique to the saliva from isoproterenol-treated animals were also adsorbed to HA. Thus alterations in proline-rich proteins of parotid saliva may influence the adherence and aggregation of oral bacteria, two processes considered important for in-vivo colonization of oral surfaces.

Influence of beta-adrenergic stimulation on glycosylation of a major, secretory N-linked glycoprotein from rat parotid salivary gland.

beta-Adrenergic stimulation with 10 microM isoproterenol increased the [3H]-mannose/[14C]-leucine ratio (three- to six-fold) of protein extracts in double-radiolabelled rat parotid acinar cells. Characteristics of oligosaccharides in a major parotid glycoprotein (Mr approximately 220,000; gp 220) were studied. Gp 220 from control and experimental cells was endoglycosidase H-insensitive, endoglycosidase F-sensitive and bound both concanavalin A and wheat germ agglutinin. Gp 220 was removed from concanavalin A-Sepharose by sequential elution with 10 mM alpha-methyl glucoside and 0.5 M alpha-methyl mannoside. These findings suggest that (1) oligosaccharides in gp 220 have both a biantennary complex and hybrid oligosaccharide chains, and (2) beta-adrenoreceptor stimulation has little effect on the gross oligosaccharide structures of this glycoprotein.

Modulation of oligosaccharide processing in an exocrine secretory glycoprotein of rat parotid cells by beta-adrenoreceptor activation.

Such stimulation of rat parotid acinar cells in vitro modulated the rate of processing of N-linked oligosaccharides in a high-molecular weight (220 kdalton) secretory glycoprotein. Conversion of polymannose-type oligosaccharides to complex-type oligosaccharides was evaluated by sensitivity to endoglucosaminidase H and alpha-mannosidase, and with a specific inhibitor of glucosidases I/II. Oligosaccharide maturation in the 220 kdalton glycoprotein required one-third to half less time in cells exposed to the beta-adrenergic agonist isoproterenol than in controls.

cAMP-mediated protein phosphorylation of microsomal membranes increases mannosylphosphodolichol synthase activity.

We have investigated the possible role of a cAMP-mediated protein-phosphorylation event(s) as the key regulatory mechanism in beta-adrenoreceptor-stimulated activation of mannosylphosphodolichol (Man-P-Dol) synthase (GDP-mannose:dolichyl-phosphate O-beta-D-mannosyltransferase, EC 2.4.1.83) in rat parotid acinar cells. Microsomal membranes isolated from these cells pretreated with 10 microM isoproterenol for 60 min showed approximately 40-80% enhanced Man-P-Dol synthase activity compared to the untreated controls. This change in enzyme activity was not associated with a significant alteration in apparent Km for GDP-mannose, but the Vmax was enhanced 2-fold. When microsomal membranes isolated from control cells were phosphorylated in vitro by a cAMP-dependent protein kinase, an increase in Man-P-Dol synthase activity, similar to that with membranes from isoproterenol-treated cells, was observed (i.e., a moderate change in Km for GDP-mannose but a 2-fold higher Vmax). Furthermore, treatment of in vitro phosphorylated microsomal membranes by alkaline phosphatase led to a substantial reduction in Man-P-Dol synthase activity. Increased Man-P-Dol synthesis (approximately 30-40%) was also observed in bovine brain and hen oviduct microsomal membranes after in vitro protein phosphorylation. In aggregate, these results strongly suggest that agents that increase cAMP in cells may modulate protein N-glycosylation in those cells by activating this key glycosyltransferase of the dolichol cascade by a cAMP-dependent protein kinase-mediated protein phosphorylation/dephosphorylation cycle.

beta-Adrenergic activation of glycosyltransferases in the dolichylmonophosphate-linked pathway of protein N-glycosylation.

beta-Adrenoreceptor stimulation of rat parotid acinar cells increases the activity of several microsomal membrane associated, dolichylmonophosphate (Dol-P) linked glycosyltransferases. The activities of Man-P-Dol synthase and Glc-P-Dol synthase are increased by approximately 50%, and the activity of N-acetylglucosaminyl 1-phosphate transferase plus N-acetylglucosaminyl transferase increased by approximately 60%, after agonist treatment. Increases in enzyme activity are (i) independent of endogenous Dol-P levels and (ii) observed under conditions in which the specific activities of donor sugar nucleotides are kept constant. Activation of these enzymes is specific since comparable levels of NADPH-cytochrome c reductase are found in control and agonist-treated membranes. The data thus provide the initial demonstration of neurotransmitter modulation of enzymes in the dolichol-linked pathway of protein N-glycosylation.

beta-Adrenergic receptor regulation of N-linked protein glycosylation in rat parotid acinar cells.

We have investigated the relationship between beta-adrenergic receptor stimulation and protein glycosylation and secretion in rat parotid gland cells in vitro. The potent beta-adrenergic agonist (-)-isoproterenol increases [3H]mannose incorporation into newly synthesized glycoproteins. This effect is enhanced if cells are first preincubated with dolichyl phosphate and is not observed after muscarinic-cholinergic or alpha-adrenergic stimulation of cells. The increase in [3H]mannose incorporation is abolished by incubation of cells with tunicamycin, suggesting that the glycosylation events being studied involved asparagine-linked oligosaccharides. The extent of increase in glycosylation is dependent on the concentration of (-)-isoproterenol to which cells are exposed. (+/-)-Propanolol totally abolishes the (-)-isoproterenol-induced increase in [3H]mannose incorporation, in a manner similar to its effects on exocrine secretion. Our findings suggest that beta-adrenergic receptor activation has a profound influence on N-linked protein glycosylation in rat parotid cells in addition to eliciting exocrine protein release.

Dolichyl phosphate supplementation increases N-linked protein glycosylation in rat parotid acinar cells without increasing glycoprotein secretion.

N-linked protein glycosylation was increased three- to five-fold, in dispersed rat parotid acinar cells in vitro, by supplementation with exogenous dolichylphosphate. Despite this increase, glycoprotein secretion from both control and dolichylphosphate-supplemented cells was comparable.

Exocrine protein secretion from human parotid glands during aging: stable release of the acidic proline-rich proteins.

Exocrine protein secretion from stimulated human parotid glands was examined in 220 adults, 20 to 88 years old. A group of parotid proteins, the anionic proline-rich proteins (PRP), was used as a marker for studying protein exocytosis. Data were evaluated as the salivary concentration of PRP (mg%), the percentage of total parotid salivary protein represented by the PRP and as total PRP output (mg/min). No differences were observed in the ability of different-aged males and females to secrete this group of exocrine secretory proteins.

Cellular characteristics of long-term cultured rat parotid acinar cells.

We have successfully maintained and biochemically characterized differentiated rat parotid acinar cells cultured for long periods (6 mo.). The cells were cultured on a reconstituted basement membrane matrix in a medium containing a variety of agents that promote cellular proliferation and differentiation. The cultured cells retain the characteristics of the parental parotid acinar cells. They exhibit both secretory granules and abundant cellular organelles required for protein synthesis and secretion. In situ hybridization and immunocytochemistry demonstrate high levels of proline-rich protein mRNA and protein, and lower levels of amylase mRNA and protein, in their cytoplasm. These findings suggest that rat parotid acinar cells can be maintained in a differentiated state in vitro for long periods, and can serve as a useful model system for studying the regulation of exocrine secretory processes.

beta-Adrenergic stimulation alters oligosaccharide pyrophosphoryl dolichol metabolism in rat parotid acinar cells.

beta-Adrenergic stimulation of rat parotid acinar cells markedly increases [3H]mannose incorporation into N-linked glycoproteins [Kousvelari, Grant, Banerjee, Newby & Baum (1984) Biochem. J. 222, 17-24]. More than 90% of this protein-bound [3H]mannose was preferentially incorporated into four secretory glycoproteins. The ratio of [3H]mannose/[14C]leucine present in these individual proteins was 1.7-4-fold greater with isoproterenol-treated cells than with untreated controls. In isoproterenol-stimulated cells, [3H]mannose incorporation into mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol was increased 2-3-fold over that observed in unstimulated cells. Similarly, formation of mannosylated oligosaccharide-PP-dolichol was increased approx. 4-fold in microsomes prepared from isoproterenol-treated cells. Also, turnover of oligosaccharide-PP-dolichol was significantly increased (5-fold) by beta-adrenergic stimulation; the half-life for oligosaccharide-PP-dolichol decreased from 6 min in control cells to 1.2 min in isoproterenol-stimulated cells. By 15 min after isoproterenol addition to acinar cells, the specific radioactivity of parotid oligosaccharide moieties increased about 3-fold over the value observed in the absence of the agonist. Taken together, these results strongly suggest that elevation of N-linked protein glycosylation in rat parotid acinar cells after beta-adrenoreceptor stimulation resulted from significant enhancement in the synthesis of mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol and the turnover of oligosaccharide-PP-dolichol.

Bromoacetylalprenololmenthane: a potent irreversible antagonist of beta adrenergic elicited protein exocytosis in rat parotid cells.

The interaction of bromoacetylalprenololmenthane (BrAlpM), an irreversible beta adrenergic receptor antagonist, with rat parotid acinar cells was studied in vitro. In the presence of BrAlpM, the rate of (-)-isoproterenol-induced exocrine secretion from cells, measured as percentage of amylase release, was markedly reduced. The concentration of (-)-isoproterenol required to elicit half-maximal protein secretion was about 100 times greater (5 microM) in the presence of 1 microM BrAlpM than in control incubations (0.05 microM). BrAlpM and propranolol were similar in their ability to inhibit parotid protein release (IC50 approximately 10(-7) M). To demonstrate that BrAlpM functioned as an irreversible beta adrenergic antagonist, cells were preincubated with BrAlpM for varying amounts of time and then washed three to six times before adding (-)-isoproterenol. At least 10 min preincubation was required to show irreversibility. Alprenolol, under the same preincubation conditions, was unable to inhibit amylase release. BrAlpM inhibited the binding of [3H]dihydroalprenolol to parotid beta adrenoreceptors over a concentration range similar to that required for inhibition of protein secretion. Cells incubated in the absence or presence of BrAlpM displayed a comparable morphologic appearance when viewed by light and electron microscopy. The degree of inhibition of isoproterenol-induced exocytosis of secretory granules by BrAlpM appeared to vary from cell to cell. These findings suggest that BrAlpM should be a useful probe to study beta adrenoreceptor function and metabolism in rat parotid acinar cells.

c-myc mRNA expression in minor salivary glands of patients with Sjögren's syndrome.

c-myc protooncogene is implicated in the pathogenesis of B cell lymphoid malignancies and high levels of c-myc mRNA expression are observed in activated blood mononuclear cells. Sjögren's syndrome (SS) is characterized by lymphocytic infiltrates of exocrine glands, remarkable B cell hyperreactivity and a strong predisposition to B cell neoplasia. In this study, c-myc protooncogene mRNA expression in 29 labial minor salivary gland biopsies from patients with primary SS and 15 controls was examined using in situ hybridization histochemistry. Two 40mer oligonucleotides from the 1st and the 2nd exon of the c-myc gene, labeled with 35S, were used as probes. To detect the origin of the cell hybridized with a c-myc probe, a combined immunochemistry in situ hybridization histochemistry technique was used. High c-myc mRNA expression was detected on acinar epithelial cells. c-myc did not correlate with c-fos and c-jun protein expression. Stronger c-myc mRNA expression was detected in labial salivary glands of patients with longer disease duration (p less than or equal to 0.002) and more intense T lymphocyte infiltrates (p less than 0.05) although these patients revealed no hypergammaglobulinemia. No correlation was observed between c-myc mRNA and B lymphocyte monoclonicity or lymphoma. In conclusion, strong c-myc mRNA expression was observed on epithelial cells of labial salivary glands from patients with primary SS. Our findings may indicate the presence of a reactivated virus hosted in these cells.