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Effect of lipids on glycoprotein sulphotransferase activity in rat submandibular salivary glands
Pergamon
0003-9969(94)00176-6
Archs oral Biol. Vol. 40, No. 5, pp. 433M.38, 1995 Copyright (~ 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0003-9969/95 $9,50 + 0.00
E F F E C T OF LIPIDS O N G L Y C O P R O T E I N S U L P H O T R A N S F E R A S E A C T I V I T Y IN RAT SUBMANDIBULAR SALIVARY GLANDS C. KASINATHAN,* S. WILLIAM, S. VAIDYANATHAN and J. LEVENTHAL Dental Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2400, U.S.A.
(Accepted 2 November 1994) Summary--Although glycoprotein sulphation has been implicated in the processing of salivary mucin, little is known about the regulation of the enzyme responsible for this event. Using desulphated glycoprotein as sulphate acceptor, the glycoprotein sulphotransferase (GPST) from Golgi membranes of submandibular salivary gland was used to study the effect of various lipids on its activity. The GPST activity in the Golgi membrane was 0.7 pmol/mg protein per rain and the activity was extractable by Triton S-100. The Km of the solubilized GPST for glycoprotein and 3'-phosphoadenosine 5'-phosphosulphate (PAPS) were 11 and 0.2/~M, respectively. Among the various lipids tested, phosphatidylinositol and sphingosine stimulated the GPST activity, while other lipids such as sphingomyelin, phosphatidylcholine and phosphatidylserine did not produce a significant effect. At 12 mol% (when expressed as mol% of sphingosine to total phospholipids plus Triton X-100) of sphingosine concentration, the enzyme activity was increased nearly 1.7-fold. The stimulatory effect of sphingosine was accompanied by a significant decrease in K m for glycoprotein from 11 to 2pM but the increase in Vm~~ was small. In contrast, the sphingosine effect did not change the Km for PAPS but increased the Vm,x nearly two fold. Of the two sphingosine analogues tested, threosphinganine and erythrosphinganine had a lesser stimulatory effect than sphingosine. Stearylamine was partially active, whereas the amino acids (glutamate, aspartate, glutamine, asparagine and serine) were not. These observations and our earlier finding of tyrosylprotein sulphotransferase inhibition by sphingosine demonstrate diverse sphingosine effects on the posttranslational sulphation involved in the processing of salivary proteins and suggest an important role for sphingosine in the regulation of salivary protein sulphation. Key words: glycoprotein sulphotransferase, tyrosylprotein sulphotransferase, submandibular salivary gland, lipids and Golgi.
proteins (Slomiany et al., 1991), information is not available on the regulation of glycoprotein Salivary glycoproteins, the major constituent of sulphotransferase. saliva, are important in the protection of the oral soft Carbohydrate sulphation of salivary glycoprotein tissues and the teeth from ulcerations, abrasions and has been characterized primarily within the Golgi caries (Levine et aL, 1985; Slomiany et al., 1986; apparatus (Murty et al., 1988). The structural Tabak et al., 1982). These protective properties of components of biological membranes, including salivary secretory proteins are acquired through post- Golgi, that house the sulphotransferase are lipids translational modifications such as sulphation and (Vance and Vance, 1988). Lipids and their metabolic acylation (Towler and Gordon, 1988; Nachman et al., products are important in cell growth and function 1986; Pauwels, Docking and Walker, 1987). The (Hannun and Bell, 1989; Majerus et al., 1988) and sulphation of the carbohydrate residues of salivary with the discovery of inhibition of protein kinase C secretory protein is catalysed by glycoprotein sulpho- by sphingosine, interest has been directed to the transferase (Slomiany et al., 1988), whereas the potential role of sphingolipid-derived products in sulphation of the tyrosine residues in these proteins cell regulation (Hannun et al., 1986). Sphingolipid is catalysed by tyrosylprotein sulphotransferase synthesis starts in the endoplasmic reticulum.by the (Sundaram el al., 1992; Kasinathan et al., 1993; condensation of serine with palmitoyl-CoA by serine Leventhal et al., 1994; Rivera et al., 1994). Although palmitoyltransferase (Mandon et al., 1992). The considerable progress has been made in under- condensation product, 3-dehydrosphinganine, is then standing the significance and physiological role of converted to D-erythrosphinganine and then to carbohydrate sulphation in the processing of salivary sphingosine. D-erythrosphinganine could also bc __ used directly for the synthesis of dihydroceramide by *To whom correspondence should be addressed, sphingosine N-acyltransferase. The sphingolipidAbbreviations: [35S]PAPS,3'-phosphoadenosine 5'-phospho- synthesizing enzymes responsible for the formation [~SS]sulphate: EAY, poly-(Glu6, Ala3Tyrt). of dihydroceramide are present in the endoplasmic INTRODUCTION
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reticulum while the latter enzymes are present in the Golgi (Futerman et al., 1990; Schwarzmann and Sandhoff, 1990). Sphingolipid bases and lys0sphingolipids, the intermediates in the biosynthesis and catabolism of sphingomyelin and glycosphingolipids, are proposed as physiological regulators of protein kinase C (Merrill and Stevens, 1989) and they also exhibit various pharmacological effects in cells (Hall et al., 1988). Recently a salivary enzyme, tyrosylprotein sulphotransferase, involved in secretory protein processing (Rivera et al., 1994), was shown to be regulated by sphingolipid bases and acidic phospholipids (Kasinathan et al., 1993). We have now investigated the effect of lipids on glycoprotein sulphotransferase, another Golgi enzyme involved in the processing of salivary protein. MATERIALS AND METHODS Male Sprague-Dawley rats, 8 weeks old with a mean weight of 175 g, were obtained from Charles River Laboratories, Wilmington, MA. [35S]PAPS (2.4 Ci/mmol) was supplied by the New England Nuclear, Boston, MA. Sphingosine, erythrosphinganine, threosphinganine, stearylamine, sphingomyelin, phosphatidycholine, phosphatidylserine and EAY (Mr 47,000) were obtained from Sigma.
Subeellular fractionation Freshly dissected rat submandibular glands were gently washed with cold 0.15 M NaC1, the fat removed and the glands weighed. They were then cut into small pieces, suspended in 5 Vol of STKM buffer (50mM tris-HC1, pH 7.4, 25 mM KCI, 5 mM Mg (CH3COO)2, 5 mM 2-mercap.toethanol, 1 mM phenylmethanesulphonyl fluoride) containing 0.25 M sucrose and homogenized in a Polytron PT-20 (Brinkman Instruments Inc.)for 10s at half-maximal speed. The resulting homogenate was again homogenized in a motor-driven Teflon homogenizer using six strokes at 300 rev/min, passed through a nylon screen, and centrifuged at 5000g for 20min. The 5000g supernatant was layered on a discontinuous gradient of 0.5 and 1.2 M sucrose in STKM buffer (Kasinathan et al., 1993). The gradients were centrifuged for 2.5 h at 25,000rev/min (90,000g) in the SW 40 rotor. The light-brown material at the 0.5 M/1.2 M sucrose interface (Golgi-rich) was collected, diluted with half-concentrated STKM buffer and centrifuged at 100,000 g for 1 h. The pellet was suspended in 10mM tris-HC1, pH 6.8, containing 20% glycerol and stored at - 7 0 ° C before use. Protein concentration was estimated by the BCA (No. 23225) protein assay reagent (Pierce Chemical Co.). Glycoprotein sulphotransferase isolation The partial purification of glycoprotein sulphotransferase was done as described for the Golgi tyrosylprotein sulphotransferase (Kasinathan et al., 1993). The submandibular Golgi membranes were suspended in 10 mM HEPES buffer, pH 7.2, containing 25% glycerol, 1 mM NaCI and 1 mM dithiothreitol. The suspension was mixed on ice for 30 min and centrifuged for 1 h at 134,000g. The resulting pellet was dissolved in t 0 m M HEPES, pH7.2,
containing 25% glycerol, 0.2mM NaCI, l mM dithiothreitol and 1%0 Triton X- 100, mixed for 30 min on ice and centrifuged as before. The supernatant was used for the glycoprotein sulphotransferase assay.
Preparation ofphospholipid suspension Sphingosine and other lipids were dried under N 2 to remove the organic solvents. After allowing the lipids to swell under vacuum for 18 h, phospholipid suspensions (1 mM or 1.3 mM) were prepared by sonication for 20 min in 10 mM MES buffer, pH 6.2. The lipid suspensions were used immediately (within 1 h)after preparation. Assay of glycoprotein sulphotransferase The glycoprotein sulphotransferase mixture contained: acceptor, 250 #g desulphated mucous glycoprotein, 2 p M [35S]PAPS, 0.5% Triton X-100, 25 mM NaF, 4 mM MgC12, 80 mM imidazole-HCl buffer at pH6.8 and 3 0 - 5 0 # g of enzyme protein in a final volume of 50 pl. Assays were initiated by the addition of the enzyme protein and run for 45 min at 37°C. Reactions were stopped by spotting 40-pl portions of the reaction mixture onto a 2.4 × 2.4 cm Whatman No. 3MM filter paper (Kasinathan et al., 1993). The papers were washed three times for 15 min in 10% trichloroacetic acid/10 mM Na2SO 4 and rinsed for 5 min in 95% ethanol. The dried papers were placed in vials containing scintillation solution and counted in a scintillation counter, TriCarb-1500. Two controis, one containing the assay mixture minus glycoprotein and the other minus enzyme protein, were employed. The glycoprotein sulphotransferase activity was expressed as pmol of [35S]-sulphate transferred to the desulphated glycoprotein per mg of enzyme protein per min and was arrived at by subtracting the total sulphation from the sulphation observed in the two controls. Of the total [35S]sulphate transferred to salivary mucin, 93% of the label was sensitive to alkaline hydrolysis, indicating carbohydrate sulphation (Huttner, 1984). Preparation of desulphated glycoprotein Deletion of the sulphate ester groups from salivary mucin was achieved by acid catalysed solvolysis (Murty et al., 1988). Glycoprotein (20 mg) was suspended in 1 ml of 0.05 M HCI in dry methanol at room temperature for 8 h. At the end of incubation the mixture was neutralized, dialysed against water and lyophilized. The desulphated glycoprotein was used as substrate for the glycoprotein sulphotransferase assay. RESULTS A subcellular distribution study on glycoprotein sulphotransferase activity in rat submandibular salivary gland had revealed that it is a Golgi membrane protein (Murty et al., 1988). Recently, in the Golgi compartment of salivary gland, we have also observed tyrosylprotein sulphotransferase activity and partially purified it to study its regulation by lipids (Kasinathan et al., 1993). Employing a similar procedure, the glycoprotein sulphotransferase activity was isolated from Golgi membranes of the submandibular salivary gland (Table 1). Purified
Glycoprotein sulphotransferase Table 1. Isolation of glycoprotein sulphotransferase activity from Golgi membranes of rat submandibular salivary gland Glycoprotein sulphotransferase Fraction (pmol/mg protein per min) Homogenate 0.06a Golgi 0.66 _+0.07 Triton X-100 supernatant 1.24 -I-0.15 Values are the mean_+SEM of three independent preparations. ~Average of duplicate determinations. Golgi membrane contained 11 times more of the activity than in the homogenate. The membraneassociated activity was solubilized using the non-ionic detergent Triton X- 100. The 100,000 g supernatant of Triton X-100-treated Golgi membrane had 21-fold higher activity than that of the homogenate. The partially purified enzyme was characterized for its substrate requirements. The effects of glycoprotein and PAPS on glycoprotein sulphotransferase activity are shown in Figs 1A and B. Glycoprotein sulphation increased with increasing concentrations of glycoprotein and PAPS. The K m value of the enzyme for glycoprotein was 1.1 mg/50 It 1. Based on the molecular weight of 2 x 10 6 reported for mucin (Slomiany et al., 1991), the Km value of 1.1 mg/50/tl observed here is equivalent to l l p M. The apparent Km value of the partially purified enzyme was the same as the value of 11/t M reported earlier for the unsolubilized enzyme (Murty et aL, 1988). The Km value of the partially purified enzyme for PAPS was 2 # M and it is lower than the value of 4 # M reported from the membrane-bound enzyme. The results in Fig. 2 show the effects of various lipids on the glycoprotein sulphotransferase activity of rat submandibular salivary gland. Among the several lipids tested, phosphatidylinositol stimulated the activity, whereas phosphatidylserine and sphingomyelin did not and rather showed a slight decrease in glycoprotein-sulphating activity at low
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concentrations. Phosphatidylcholine exhibited a slight stimulation in sulphation at 300/~ M. We also investigated the effect of sphingosine on glycoprotein sulphotransferase activity (Fig. 3), which it stimulated in a concentration-dependent manner. At 12mol% of sphingosine the stimulation in activity was around 1.7-fold. The assay medium used for measuring this activity contained 1400 # M Triton X-100 and the Goigi membrane contained 924 # M of phospholipid. Thus, 300 p M sphingosine
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Fig. 1. Effect of glycoprotein and psS]PAPS concentration on the activity of solubilized submandibular glycoprotein sulphotransferase. (A) The activity was measured with varying glycoprotein concentrations; (B) the enzyme was assayed at different PAPS concentrations. Reactions were carried out for 45 rain at 37°C and processed for the quantitation of sulphation. AOB
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C. Kasinathan et al. also investigated; its effect on the activity at varying glycoprotein and PAPS concentrations is shown in Fig. 4. When the glycoprotein concentration was increased against a fixed PAPS concentration, sphingosine decreased the K m value for glycoprotein from 11 to 2 # M (Fig. 4A); the Vmax remained nearly unchanged. When the PAPS concentration was increased against a fixed glycoprotein concentration, sphingosine increased both Vmax (1.1-2.2pmol/mg per min) and, to a lesser extent, the K m value for PAPS (from 2.0 to 3.3 #m) (Fig. 4B).
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in the assay is equivalent to 12 mol%. In a separate experiment the specificity of the sphingosine stimulation of glycoprotein sulphotransferase was determined by studying the effect of sphingosine analogues such as threo- and erythrosphinganine, which also stimulated the activity but to a lesser extent. To determine if the stimulation of glycoprotein sulphotransferase was attributable solely to the presence of a free amino group, the effect of stearylamine and amino acids (Table 2) on its activity was determined; stearylamine stimulated the activity but to lesser extent than sphingosine. Amino acids glutamate, aspartate, glutamine, asparagine and serine caused no increase in glycoprotein sulphotransferase activity. These results suggest that the structural requirement for glycoprotein sulphotransferase stimulation is a free amino group and a long alkyl chain, The mechanism involved in the stimulation of glycoprotein sulphotransferase by sphingosine was Table 2. Effect of sphingosine, stearylamine and amino acids on glycoprotein sulphotransferase activity in rat submandibular salivary glands Lipid Glycoprotein sulphotransferase (200pM) (%control) Sphingosine 160+4 a Stearylamine 113 _+2" Glutamate 93 Aspartate 96 Glutamine 95 Asparagine 94 Serine 96 One hundred % activity is 0.93 pmol/mg per min, Values are the average of duplicate determinations. "Values are mean +SEM of three independent determinations.
We have described to the best of our knowledge, for the first time, the influence of various lipids on glycoprotein sulphotransferase activity in submandibular salivary glands. Among the several lipids tested on this activity, sphingosine and phosphatidylinositol were the most potent stimulators. Previously, sphingosine and phosphatidylinositol have been shown to regulate the tyrosylprotein sulphotransferase, a salivary enzyme, implicated in the processing of salivary proteins (Kasinathan et al., 1993; Leventhal et al., 1994; Rivera et al., 1994). The sphingosine concentration required for the stimulation of glycoprotein sulphotransferase (190%) was approx. 12 mol%; and 30% stimulation was achieved by 6 mol%, a concentration range known to inhibit tyrosylprotein sulphotransferase activity in salivary glands (Kasinathan et al., 1993) and also protein kinase C activity in platelets (Hannun et al., 1986). The concentration at which sphingosine regulates the function of these and other enzymes varies from 5 to 25 mol% (Hannun et al., 1986; Sohal and Cornell, 1990). Several other studies have demonstrated the activation by sphingosine of casein kinase (McDonald et al., 1991), epidermal growth factorreceptor kinase (Faucher et al., 1988), and phospholipase D (Kiss and Andersen, 1990). In the case of casein kinase, the activation is largely due to an increase in the enzyme's affinity for its protein substrate ( M c D o n a l d etal., 1991). Similarly, we have observed an increase in the affinity of glycoprotein sulphotransferase for glycoprotein in the presence of sphingosine. In contrast to the above the salivary tyrosylprotein sulphotransferase was inhibited by sphingosine (Kasinathan et al., 1993). However, the effect of sphingosine on both carbohydrate and tyrosine sulphation in salivary gland had one similarity in that it was exerted by altering the Km values of the glycoprotein and tyrosylprotein sulphotransferase for their respective substrates, glycoprotein and EAY. The activation of glycoprotein sulphotransferase by sphingosine displayed structural selectivity in that erythrosphinganine and threosphinganine were less effective stimulators than sphingosine. Furthermore, stearylamine stimulated the glycoprotein sulphotransferase activity but not the amino acids, suggesting that the stimulation is related not only to the presence of a free amino group but also to the length of the hydrocarbon chain, which was also the requirement for the inhibition of tyrosin e sulphation in salivary gland (Kasinathan et al., 1993). Polyamines exhibited a similar stimulatory effect
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Fig. 4. Effect of sphingosine on glycoprotein sulphotransferase activity at varying substrate concentrations. Increasing concentrations of glycoprotein (A) and [35S]PAPS (B) were added to the assay medium with 150 #M sphingosine (Q) or without sphingosine (©). on serum glycosaminoglycan sulphotransferase, an enzyme closely related to glycoprotein sulphotransferase (Sugahara et al., 1989). The activation of glycosaminoglycan sulphotransferase was due to a decreases in the K m values of the enzyme for PAPS. In contrast to that finding, the activation of salivary glycoprotein sulphotransferase by sphingosine resulted from a decrease in the K m values of the enzyme for glycoprotein rather than for PAPS. There is considerable volume of literature indicating a close relation between cell function and lipid metabolism (Hannun and Bell, 1989; Majerus et al., 1988). Our present results and our earlier report (Kasinathan et al., 1993) suggest that, in salivary glands, sphingosine and phosphatidylinositol may regulate the sulphation events responsible for the processing of salivary protein. There have also been a number of studies describing changes in the levels of phospholipids in dental caries and other diseases (Aono et al., 1982; Murty et al., 1982; Slomiany, Murty and Slomiany, 1985; Somiany et al., 1986). Although it is difficult to say at this time whether the lipid-regulated changes in sulphotransferase activities are associated with altered salivary sulphoprotein metabolism, there are reports of the significance of carbohydrate sulphation in the formation of high molecular-weight mucin (Slomiany et al., 1991) and of tyrosine sulphation in protein secretion (Huttner, 1982; Huttner, 1988). The ability of sphingosine and phosphatidylinositol to regulate carbohydrate and tyrosine sulphation (Kasinathan et al., 1993), the two processes necessary for the processing of salivary proteins, provides a powerful tool for exploring the mechanisms of signal transduction. This work was supported by National Institute of Dental Research Grants DE10538 and DE09874. Acknowledgements
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Levine M. J., Tabak L. A., Reddy M. and Mandel I . D . (1985) Nature of salivary pellicles in microbial adherence: role of salivary mucins. In Molecular Basis o f Oral Microbial Adhesion (Eds Mergenhagen S. E. and Rosan B.), pp. 125-30. Am. Soc. Microbiol., Washington, D.C. Majerus P. W., Connolly T. M., Bansal V. S., Inhorn C. R., Ross T. S. and Lips D. L. (1988) Inositol phosphates: synthesis and degradation. J. biol. Chem. 263, 3051-3054. Mandon E. C., Ehses I., Rother J., Echten G . V . and Sandhoff K. (1992) Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine Nacyltransferase in mouse liver. J. biol. Chem. 267, 11144-11148. McDonald O. B., Hannun Y. A., Reynolds C. Y. and Sahyoun N. (1991) Activation of casein kinase II by sphingosine. J. biol. Chem. 266, 21773 21776. Merrill A. H. and Stevens V. L. (1989) Modulation of protein kinase C and diverse cell functions by sphingosine--a pharmacologically interesting compound linking sphingolipids and signal transduction. Biochim. biophys. Acta 1010, 131 139. Murty V. L. N., Slomiany B. L., Zdebska E., Slomiany A. and Mandel I. D. (1982) Salivary lipids in caries resistant individuals. I R C S med. Sci 10, 359. Murty V. L. N., Bilski J., Liau Y. H., Slomiany A. and Slomiany B. (1988) Sulfation in vitro of mucous glycoprotein by submandibular salivary gland: effects of prostaglandin and acetylsalicylic acid. Biochim. biophys. Acta 996, 287 296. Nachman R. J., Holman G. M., Cook B. J., Haddon W. F. and Ling N. (1986) Leukosulfakinin-11, a blocked sulfated insert neuro peptide with homology to cholecystokinin and gastrin. Biochem. biophys. Res. Commun. 140, 357-364. Pauwels S., Dockray G. J. and Walker R. (1987) Comparison of the metabolism of sulfated and unsulfated heptadecapeptidegastrininhumans. Gastroenterology92, 1220 1225. Rivera T., Macedo B., Slomiany B. L., Slomiany A. and
Kasinathan C. (1994) Presence of tyrosylprotein sulfates in rat saliva. J. Dent. Res. 73, 151. Schwarzmann G. and Sandhoff K. (1990) Metabolism and intracellular transport ofglycosphingolpids. Biochemistry 29, 10865-10871. Sohal P. S. and Cornell B. (1990) Sphingosine inhibits the activity of rat liver CTP phosphocholine cytidyltransferase. J. biol. Chem. 265, 11746-11750. Slomiany B. L., Murty V. L. N. and Slomiany A. (1985) Salivary lipids in health and disease. Prog. lipid Res. 24, 311-324. Slomiany B. L., Murty V. L. N., Slomiany A., Zielenski J. and Mandel I. D. (1986) Mucous glycoprotein of human saliva: differences in the associated and covalently bound lipids and caries. Biochim. Biophys. Acta 881, 18 28. Slomiany A., Murty V. L. N., Liau Y. H., Carter S. R. and Slomiany B. L. (1988)Enzymatic sulfation of mucus glycoprotein in rat sublingual salivary gland. Archs oral Biol. 33, 669 676. Slomiany B. L., Liau Y. H., Li Q., Fekete Z. and Slomiany A. (1991) Role of sulfation in posttranslational processing of rat salivary mucins. Archs oral Biol. 36, 785-790. Sugahara K., Nakamura M., Nagisa J., Masuda M., Nunokawa Y., Fujii N. and Yamashina I. (1989) Regulation of serum glycosaminoglycan sulfotransferase activities: Inhibition by sulfated glycosaminoglycans and activation by polyamines and basic peptides including a polylysine-containing segment of the c-Ki-ras 2 protein. J. Biochem. 106, 910 919. Sundaram P., Slomiany A., Slomiany B. L. and Kasinathan C. (1992) Tyrosylprotein sulfotransferase in rat submandibular salivary glands. Int. J. Biochem 24, 663 667. Tabak L. A., Levine M. J., Mandel I. D. and Ellison S. A. (1982) Role of salivary mucin in the protection of the oral cavity. J. oral Pathol. 11, 1 17. Towler D. A. and Gordon J. I. (1988) The biology and enzymology of eukaryotic protein acylation. Ann. rev. Biochem. 57, 69-99. Vance J. E. and Vance D. E. (1988) Does rat liver Golgi have the capacity of synthesize phospholipids for lipoprotein secretion? J. biol. Chem. 263, 5898-5909.
0003-9969(94)00176-6
Archs oral Biol. Vol. 40, No. 5, pp. 433M.38, 1995 Copyright (~ 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0003-9969/95 $9,50 + 0.00
E F F E C T OF LIPIDS O N G L Y C O P R O T E I N S U L P H O T R A N S F E R A S E A C T I V I T Y IN RAT SUBMANDIBULAR SALIVARY GLANDS C. KASINATHAN,* S. WILLIAM, S. VAIDYANATHAN and J. LEVENTHAL Dental Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2400, U.S.A.
(Accepted 2 November 1994) Summary--Although glycoprotein sulphation has been implicated in the processing of salivary mucin, little is known about the regulation of the enzyme responsible for this event. Using desulphated glycoprotein as sulphate acceptor, the glycoprotein sulphotransferase (GPST) from Golgi membranes of submandibular salivary gland was used to study the effect of various lipids on its activity. The GPST activity in the Golgi membrane was 0.7 pmol/mg protein per rain and the activity was extractable by Triton S-100. The Km of the solubilized GPST for glycoprotein and 3'-phosphoadenosine 5'-phosphosulphate (PAPS) were 11 and 0.2/~M, respectively. Among the various lipids tested, phosphatidylinositol and sphingosine stimulated the GPST activity, while other lipids such as sphingomyelin, phosphatidylcholine and phosphatidylserine did not produce a significant effect. At 12 mol% (when expressed as mol% of sphingosine to total phospholipids plus Triton X-100) of sphingosine concentration, the enzyme activity was increased nearly 1.7-fold. The stimulatory effect of sphingosine was accompanied by a significant decrease in K m for glycoprotein from 11 to 2pM but the increase in Vm~~ was small. In contrast, the sphingosine effect did not change the Km for PAPS but increased the Vm,x nearly two fold. Of the two sphingosine analogues tested, threosphinganine and erythrosphinganine had a lesser stimulatory effect than sphingosine. Stearylamine was partially active, whereas the amino acids (glutamate, aspartate, glutamine, asparagine and serine) were not. These observations and our earlier finding of tyrosylprotein sulphotransferase inhibition by sphingosine demonstrate diverse sphingosine effects on the posttranslational sulphation involved in the processing of salivary proteins and suggest an important role for sphingosine in the regulation of salivary protein sulphation. Key words: glycoprotein sulphotransferase, tyrosylprotein sulphotransferase, submandibular salivary gland, lipids and Golgi.
proteins (Slomiany et al., 1991), information is not available on the regulation of glycoprotein Salivary glycoproteins, the major constituent of sulphotransferase. saliva, are important in the protection of the oral soft Carbohydrate sulphation of salivary glycoprotein tissues and the teeth from ulcerations, abrasions and has been characterized primarily within the Golgi caries (Levine et aL, 1985; Slomiany et al., 1986; apparatus (Murty et al., 1988). The structural Tabak et al., 1982). These protective properties of components of biological membranes, including salivary secretory proteins are acquired through post- Golgi, that house the sulphotransferase are lipids translational modifications such as sulphation and (Vance and Vance, 1988). Lipids and their metabolic acylation (Towler and Gordon, 1988; Nachman et al., products are important in cell growth and function 1986; Pauwels, Docking and Walker, 1987). The (Hannun and Bell, 1989; Majerus et al., 1988) and sulphation of the carbohydrate residues of salivary with the discovery of inhibition of protein kinase C secretory protein is catalysed by glycoprotein sulpho- by sphingosine, interest has been directed to the transferase (Slomiany et al., 1988), whereas the potential role of sphingolipid-derived products in sulphation of the tyrosine residues in these proteins cell regulation (Hannun et al., 1986). Sphingolipid is catalysed by tyrosylprotein sulphotransferase synthesis starts in the endoplasmic reticulum.by the (Sundaram el al., 1992; Kasinathan et al., 1993; condensation of serine with palmitoyl-CoA by serine Leventhal et al., 1994; Rivera et al., 1994). Although palmitoyltransferase (Mandon et al., 1992). The considerable progress has been made in under- condensation product, 3-dehydrosphinganine, is then standing the significance and physiological role of converted to D-erythrosphinganine and then to carbohydrate sulphation in the processing of salivary sphingosine. D-erythrosphinganine could also bc __ used directly for the synthesis of dihydroceramide by *To whom correspondence should be addressed, sphingosine N-acyltransferase. The sphingolipidAbbreviations: [35S]PAPS,3'-phosphoadenosine 5'-phospho- synthesizing enzymes responsible for the formation [~SS]sulphate: EAY, poly-(Glu6, Ala3Tyrt). of dihydroceramide are present in the endoplasmic INTRODUCTION
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reticulum while the latter enzymes are present in the Golgi (Futerman et al., 1990; Schwarzmann and Sandhoff, 1990). Sphingolipid bases and lys0sphingolipids, the intermediates in the biosynthesis and catabolism of sphingomyelin and glycosphingolipids, are proposed as physiological regulators of protein kinase C (Merrill and Stevens, 1989) and they also exhibit various pharmacological effects in cells (Hall et al., 1988). Recently a salivary enzyme, tyrosylprotein sulphotransferase, involved in secretory protein processing (Rivera et al., 1994), was shown to be regulated by sphingolipid bases and acidic phospholipids (Kasinathan et al., 1993). We have now investigated the effect of lipids on glycoprotein sulphotransferase, another Golgi enzyme involved in the processing of salivary protein. MATERIALS AND METHODS Male Sprague-Dawley rats, 8 weeks old with a mean weight of 175 g, were obtained from Charles River Laboratories, Wilmington, MA. [35S]PAPS (2.4 Ci/mmol) was supplied by the New England Nuclear, Boston, MA. Sphingosine, erythrosphinganine, threosphinganine, stearylamine, sphingomyelin, phosphatidycholine, phosphatidylserine and EAY (Mr 47,000) were obtained from Sigma.
Subeellular fractionation Freshly dissected rat submandibular glands were gently washed with cold 0.15 M NaC1, the fat removed and the glands weighed. They were then cut into small pieces, suspended in 5 Vol of STKM buffer (50mM tris-HC1, pH 7.4, 25 mM KCI, 5 mM Mg (CH3COO)2, 5 mM 2-mercap.toethanol, 1 mM phenylmethanesulphonyl fluoride) containing 0.25 M sucrose and homogenized in a Polytron PT-20 (Brinkman Instruments Inc.)for 10s at half-maximal speed. The resulting homogenate was again homogenized in a motor-driven Teflon homogenizer using six strokes at 300 rev/min, passed through a nylon screen, and centrifuged at 5000g for 20min. The 5000g supernatant was layered on a discontinuous gradient of 0.5 and 1.2 M sucrose in STKM buffer (Kasinathan et al., 1993). The gradients were centrifuged for 2.5 h at 25,000rev/min (90,000g) in the SW 40 rotor. The light-brown material at the 0.5 M/1.2 M sucrose interface (Golgi-rich) was collected, diluted with half-concentrated STKM buffer and centrifuged at 100,000 g for 1 h. The pellet was suspended in 10mM tris-HC1, pH 6.8, containing 20% glycerol and stored at - 7 0 ° C before use. Protein concentration was estimated by the BCA (No. 23225) protein assay reagent (Pierce Chemical Co.). Glycoprotein sulphotransferase isolation The partial purification of glycoprotein sulphotransferase was done as described for the Golgi tyrosylprotein sulphotransferase (Kasinathan et al., 1993). The submandibular Golgi membranes were suspended in 10 mM HEPES buffer, pH 7.2, containing 25% glycerol, 1 mM NaCI and 1 mM dithiothreitol. The suspension was mixed on ice for 30 min and centrifuged for 1 h at 134,000g. The resulting pellet was dissolved in t 0 m M HEPES, pH7.2,
containing 25% glycerol, 0.2mM NaCI, l mM dithiothreitol and 1%0 Triton X- 100, mixed for 30 min on ice and centrifuged as before. The supernatant was used for the glycoprotein sulphotransferase assay.
Preparation ofphospholipid suspension Sphingosine and other lipids were dried under N 2 to remove the organic solvents. After allowing the lipids to swell under vacuum for 18 h, phospholipid suspensions (1 mM or 1.3 mM) were prepared by sonication for 20 min in 10 mM MES buffer, pH 6.2. The lipid suspensions were used immediately (within 1 h)after preparation. Assay of glycoprotein sulphotransferase The glycoprotein sulphotransferase mixture contained: acceptor, 250 #g desulphated mucous glycoprotein, 2 p M [35S]PAPS, 0.5% Triton X-100, 25 mM NaF, 4 mM MgC12, 80 mM imidazole-HCl buffer at pH6.8 and 3 0 - 5 0 # g of enzyme protein in a final volume of 50 pl. Assays were initiated by the addition of the enzyme protein and run for 45 min at 37°C. Reactions were stopped by spotting 40-pl portions of the reaction mixture onto a 2.4 × 2.4 cm Whatman No. 3MM filter paper (Kasinathan et al., 1993). The papers were washed three times for 15 min in 10% trichloroacetic acid/10 mM Na2SO 4 and rinsed for 5 min in 95% ethanol. The dried papers were placed in vials containing scintillation solution and counted in a scintillation counter, TriCarb-1500. Two controis, one containing the assay mixture minus glycoprotein and the other minus enzyme protein, were employed. The glycoprotein sulphotransferase activity was expressed as pmol of [35S]-sulphate transferred to the desulphated glycoprotein per mg of enzyme protein per min and was arrived at by subtracting the total sulphation from the sulphation observed in the two controls. Of the total [35S]sulphate transferred to salivary mucin, 93% of the label was sensitive to alkaline hydrolysis, indicating carbohydrate sulphation (Huttner, 1984). Preparation of desulphated glycoprotein Deletion of the sulphate ester groups from salivary mucin was achieved by acid catalysed solvolysis (Murty et al., 1988). Glycoprotein (20 mg) was suspended in 1 ml of 0.05 M HCI in dry methanol at room temperature for 8 h. At the end of incubation the mixture was neutralized, dialysed against water and lyophilized. The desulphated glycoprotein was used as substrate for the glycoprotein sulphotransferase assay. RESULTS A subcellular distribution study on glycoprotein sulphotransferase activity in rat submandibular salivary gland had revealed that it is a Golgi membrane protein (Murty et al., 1988). Recently, in the Golgi compartment of salivary gland, we have also observed tyrosylprotein sulphotransferase activity and partially purified it to study its regulation by lipids (Kasinathan et al., 1993). Employing a similar procedure, the glycoprotein sulphotransferase activity was isolated from Golgi membranes of the submandibular salivary gland (Table 1). Purified
Glycoprotein sulphotransferase Table 1. Isolation of glycoprotein sulphotransferase activity from Golgi membranes of rat submandibular salivary gland Glycoprotein sulphotransferase Fraction (pmol/mg protein per min) Homogenate 0.06a Golgi 0.66 _+0.07 Triton X-100 supernatant 1.24 -I-0.15 Values are the mean_+SEM of three independent preparations. ~Average of duplicate determinations. Golgi membrane contained 11 times more of the activity than in the homogenate. The membraneassociated activity was solubilized using the non-ionic detergent Triton X- 100. The 100,000 g supernatant of Triton X-100-treated Golgi membrane had 21-fold higher activity than that of the homogenate. The partially purified enzyme was characterized for its substrate requirements. The effects of glycoprotein and PAPS on glycoprotein sulphotransferase activity are shown in Figs 1A and B. Glycoprotein sulphation increased with increasing concentrations of glycoprotein and PAPS. The K m value of the enzyme for glycoprotein was 1.1 mg/50 It 1. Based on the molecular weight of 2 x 10 6 reported for mucin (Slomiany et al., 1991), the Km value of 1.1 mg/50/tl observed here is equivalent to l l p M. The apparent Km value of the partially purified enzyme was the same as the value of 11/t M reported earlier for the unsolubilized enzyme (Murty et aL, 1988). The Km value of the partially purified enzyme for PAPS was 2 # M and it is lower than the value of 4 # M reported from the membrane-bound enzyme. The results in Fig. 2 show the effects of various lipids on the glycoprotein sulphotransferase activity of rat submandibular salivary gland. Among the several lipids tested, phosphatidylinositol stimulated the activity, whereas phosphatidylserine and sphingomyelin did not and rather showed a slight decrease in glycoprotein-sulphating activity at low
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concentrations. Phosphatidylcholine exhibited a slight stimulation in sulphation at 300/~ M. We also investigated the effect of sphingosine on glycoprotein sulphotransferase activity (Fig. 3), which it stimulated in a concentration-dependent manner. At 12mol% of sphingosine the stimulation in activity was around 1.7-fold. The assay medium used for measuring this activity contained 1400 # M Triton X-100 and the Goigi membrane contained 924 # M of phospholipid. Thus, 300 p M sphingosine
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in the assay is equivalent to 12 mol%. In a separate experiment the specificity of the sphingosine stimulation of glycoprotein sulphotransferase was determined by studying the effect of sphingosine analogues such as threo- and erythrosphinganine, which also stimulated the activity but to a lesser extent. To determine if the stimulation of glycoprotein sulphotransferase was attributable solely to the presence of a free amino group, the effect of stearylamine and amino acids (Table 2) on its activity was determined; stearylamine stimulated the activity but to lesser extent than sphingosine. Amino acids glutamate, aspartate, glutamine, asparagine and serine caused no increase in glycoprotein sulphotransferase activity. These results suggest that the structural requirement for glycoprotein sulphotransferase stimulation is a free amino group and a long alkyl chain, The mechanism involved in the stimulation of glycoprotein sulphotransferase by sphingosine was Table 2. Effect of sphingosine, stearylamine and amino acids on glycoprotein sulphotransferase activity in rat submandibular salivary glands Lipid Glycoprotein sulphotransferase (200pM) (%control) Sphingosine 160+4 a Stearylamine 113 _+2" Glutamate 93 Aspartate 96 Glutamine 95 Asparagine 94 Serine 96 One hundred % activity is 0.93 pmol/mg per min, Values are the average of duplicate determinations. "Values are mean +SEM of three independent determinations.
We have described to the best of our knowledge, for the first time, the influence of various lipids on glycoprotein sulphotransferase activity in submandibular salivary glands. Among the several lipids tested on this activity, sphingosine and phosphatidylinositol were the most potent stimulators. Previously, sphingosine and phosphatidylinositol have been shown to regulate the tyrosylprotein sulphotransferase, a salivary enzyme, implicated in the processing of salivary proteins (Kasinathan et al., 1993; Leventhal et al., 1994; Rivera et al., 1994). The sphingosine concentration required for the stimulation of glycoprotein sulphotransferase (190%) was approx. 12 mol%; and 30% stimulation was achieved by 6 mol%, a concentration range known to inhibit tyrosylprotein sulphotransferase activity in salivary glands (Kasinathan et al., 1993) and also protein kinase C activity in platelets (Hannun et al., 1986). The concentration at which sphingosine regulates the function of these and other enzymes varies from 5 to 25 mol% (Hannun et al., 1986; Sohal and Cornell, 1990). Several other studies have demonstrated the activation by sphingosine of casein kinase (McDonald et al., 1991), epidermal growth factorreceptor kinase (Faucher et al., 1988), and phospholipase D (Kiss and Andersen, 1990). In the case of casein kinase, the activation is largely due to an increase in the enzyme's affinity for its protein substrate ( M c D o n a l d etal., 1991). Similarly, we have observed an increase in the affinity of glycoprotein sulphotransferase for glycoprotein in the presence of sphingosine. In contrast to the above the salivary tyrosylprotein sulphotransferase was inhibited by sphingosine (Kasinathan et al., 1993). However, the effect of sphingosine on both carbohydrate and tyrosine sulphation in salivary gland had one similarity in that it was exerted by altering the Km values of the glycoprotein and tyrosylprotein sulphotransferase for their respective substrates, glycoprotein and EAY. The activation of glycoprotein sulphotransferase by sphingosine displayed structural selectivity in that erythrosphinganine and threosphinganine were less effective stimulators than sphingosine. Furthermore, stearylamine stimulated the glycoprotein sulphotransferase activity but not the amino acids, suggesting that the stimulation is related not only to the presence of a free amino group but also to the length of the hydrocarbon chain, which was also the requirement for the inhibition of tyrosin e sulphation in salivary gland (Kasinathan et al., 1993). Polyamines exhibited a similar stimulatory effect
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Fig. 4. Effect of sphingosine on glycoprotein sulphotransferase activity at varying substrate concentrations. Increasing concentrations of glycoprotein (A) and [35S]PAPS (B) were added to the assay medium with 150 #M sphingosine (Q) or without sphingosine (©). on serum glycosaminoglycan sulphotransferase, an enzyme closely related to glycoprotein sulphotransferase (Sugahara et al., 1989). The activation of glycosaminoglycan sulphotransferase was due to a decreases in the K m values of the enzyme for PAPS. In contrast to that finding, the activation of salivary glycoprotein sulphotransferase by sphingosine resulted from a decrease in the K m values of the enzyme for glycoprotein rather than for PAPS. There is considerable volume of literature indicating a close relation between cell function and lipid metabolism (Hannun and Bell, 1989; Majerus et al., 1988). Our present results and our earlier report (Kasinathan et al., 1993) suggest that, in salivary glands, sphingosine and phosphatidylinositol may regulate the sulphation events responsible for the processing of salivary protein. There have also been a number of studies describing changes in the levels of phospholipids in dental caries and other diseases (Aono et al., 1982; Murty et al., 1982; Slomiany, Murty and Slomiany, 1985; Somiany et al., 1986). Although it is difficult to say at this time whether the lipid-regulated changes in sulphotransferase activities are associated with altered salivary sulphoprotein metabolism, there are reports of the significance of carbohydrate sulphation in the formation of high molecular-weight mucin (Slomiany et al., 1991) and of tyrosine sulphation in protein secretion (Huttner, 1982; Huttner, 1988). The ability of sphingosine and phosphatidylinositol to regulate carbohydrate and tyrosine sulphation (Kasinathan et al., 1993), the two processes necessary for the processing of salivary proteins, provides a powerful tool for exploring the mechanisms of signal transduction. This work was supported by National Institute of Dental Research Grants DE10538 and DE09874. Acknowledgements
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