Biochimica et Biophysica Acta, 439 (1976) 26-37 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37374
F U R T H E R STUDIES ON A H I G H L Y P U R I F I E D G L Y C O P R O T E I N FROM T H E I N T I M A L R E G I O N OF PORCINE AORTA
B R U C E I. ROBERTS and P R E M A N A N D V. W A G H
Connective Tissue Laboratory, Veterans Administration Hospital, 300 E. Roosevelt Road, Little Rock, Ark. 72206 (U.S.A.) (Received December 17th, 1975)
SUMMARY
Highly purified glycoprotein from the intimal region of porcine aorta was isolated with minor modifications of the procedure described previously. The molecular weight of the glycoprotein as determined by sedimentation equilibrium method either in presence of 0.1 M NaCI or 6 M guanidine.HC1 containing fl-mercaptoethanol was 72 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native glycoprotein and its S-carboxyamidomethyl derivative at different acrylamide concentrations showed no difference in the molecular weight indicating the absence of subunits. Attempts to determine the identity of the amino-terminal acid by a dansylation technique indicated that the amino group is not flee. The carboxy-terminal amino acid was found to be serine after treatment of the glycoprotein with carboxypeptidase A. The glycoprotein did not contain an alkali-labile (O-glycosidic) carbohydrate-protein linkage as tested by the fl-elimination reaction. The release of monosaccharides from the glycoprotein as a function of time was studied employing mild acid hydrolysis (0.5 M HCI, 80 °C) and also by the use of neuraminidase, tt-Dand fl-D-glucosidases and fl-D-N-acetylglucosaminidase. From the observations on the release of monosaccharides and analogy with standard features determined by other investigators on soluble aortic glycoproteins, a prediction has been made as to the general features of the carbohydrate moiety of the glycoprotein.
INTRODUCTION
Mammalian arterial wall contains a variety of glycoproteins, some of which can be readily extracted with neutral buffers. Several soluble glycoproteins, as distinct from those which require more rigorous conditions for extraction, e.g. the use of trichloroacetic acid, high alkaline pH or urea, have been purified from bovine aorta [1, 2]. Although structural studies on the carbohydrate component of these glycoproteins have been attempted [2, 3], very little is as yet known regarding the nature of the carbohydrate moiety of glycoproteins from the aorta. We have previously reported a procedure for the isolation of a novel glycoprotein from the intimal region of porcine aorta [4]. The intimal glycoprotein contained 4 mol of hexosamine, 3 mol each of glucose and galactose, 2 mol of mannose
27 and 1 mol each of fucose and sialic acid per mol of glycoprotein (molecular weight 72 000). We report here some of the molecular properties of the glycoprotein and propose a tentative structure for the carbohydrate component consistent with the experimental results. MATERIALS AND METHODS Chemicals were obtained as follows: DEAE-cellulose (Cellex D, 0.7 mequiv./g), Coomassie brilliant blue R-250 dye and Bio-Gel P-150, Bio-Rad; Sephadex G-25 and G-15, Pharmacia Fine Chemicals; bovine pancreatic carboxypeptidase A, porcine pancreatic carboxypeptidase B, neuraminidase (Clostridium perfringens), and Dnsamino acids, Sigma Chemical Co. ; fl-o-glucosidase (sweet almond) and t~-o-glucosidase (yeast), Boehringer Mannheim Corp. All other chemicals were of reagent grade. fl-o-N-Acetylhexosaminidase (Jack bean) and bovine submaxillary mucin were gifts of Dr. Y. T. Li and Dr. Ward Pigman, respectively. Thoracic aortae from freshly killed pigs (about 6 months of age) were obtained from a local abattoir. The intima powder was prepared by the procedures previously described [4, 5]. Isolation of porcine intimal glycoprotein. The procedure for the isolation of glycoprotein described previously [4] was used with some modifications. In the present study, instead of chromatography on the first DEAE-cellulose column, a batchwise DEAE-cellulose fractionation method was used allowing us to process larger amounts of tissue. The dialyzed (NH4)2SO4 precipitate solution was added dropwise to a 1:1 (v/v) slurry of DEAE-cellulose in 0.005 M Tris.HCl buffer containing 0.001 M EDTA, pH 7.0 (standard buffer) under constant stirring. (A ratio of 2 ml packed resin per mg protein was necessary for maximal adsorption of the glycoprotein). The slurry was stirred for 3 h. The resin collected by centrifugation (1020 × g, 30 rain) was resuspended in an equal volume of standard buffer and stirred as above. After centrifugation, the supernatant fraction was discarded. Thereafter, the resin was stirred in an equal volume of standard buffer containing 0.2 M NaC1 for 3 h and centrifuged. This 0.2 M NaC1 elution was repeated two more times. The supernatant fractions from the three centrifugations were combined, dialyzed against standard buffer and lyophilized. The lyophilized product was dissolved in a small amount of standard buffer and dialyzed exhaustively against the same buffer. The solution was then applied to the second DEAE-cellulose column essentially as described previously [4]. The protein eluted in the region of the gradient corresponding to 0.16-0.18 M NaC1 contained the intimal glycoprotein. Small amounts of contaminating proteins remained due to these modifications. The contaminants were removed by passage of the glycoprotein fraction through a column of Bio-Gel P-150. Elution was performed with the standard buffer. Effluent from the column was monitored for protein at 280 nm employing an automatic ultraviolet analyzer. A major protein peak, eluting soon after the void volume of the column, was pooled, dialyzed against deionized water and lyophilized. This fraction was homogeneous as observed by disc gel electrophoresis and was identical in composition to the intimal glycoprotein reported previously [4]. Results from six different experiments were reproducible. The purity of the glycoprotein was tested by electrophoresis on polyacrytamide
28 gels. Electrophoresis was performed in 7-7.5 ~ gels using Tris/glycine buffer, pH 8.4 [6], and in 7.5~o gels using 0.1 M sodium phosphate buffer, pH 7.2, containing 0.1 sodium dodecyl sulfate [7]. Gels were stained and destained [8] using Coomassie brilliant blue R-250 dye. Analytical methods. Analysis of individual neutral sugars released from the glycoprotein by acid hydrolysis or by the action of glycosidases was accomplished by gas-liquid chromatography of the alditol acetates [9, 10]. Sialic acid was determined by the thiobarbituric acid method [11]. Hexosamine was quantitated by the procedure of Gatt and Berman [12]. Glucosamine was identified as the sole amino sugar by use of a Beckman 120C amino acid analyzer following hydrolysis of the glycoprotein in 2 M HCI for 16 h at 100 °C. The method of Reissig et al. [13] was used for N-acetyl amino sugar analysis. Other analytical procedures have been previously described [4]. Molecular weight determhration. The molecular weight of porcine intimal glycoprotein was determined both by sedimentation equilibrium method [14] using a Beckman Model E ultracentrifuge and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [7, 15]. In sedimentation equilibrium studies, the glycoprotein was dissolved in 0.1 M NaC1 and in 6 M guanidine.HCl containing 0.1~o flmercaptoethanol. Mixtures containing porcine intimal glycoprotein and standard proteins of known molecular weights were electrophoresed on 5, 7.5 and 10~ polyacrylamide gels in 0.1 M sodium phosphate buffer at pH 7.2 containing 1 ~ sodium dodecyi sulfate. Electrophoresis was performed at a current of 3.3-4 mA (25 oC) per tube for 1.5-5 h. In a separate experiment, the glycoprotein was first reduced with/3mercaptoethanol in 6 M urea and then alkylated with iodoacetamide [16]. The Scarboxyarnidomethylated derivative was electrophoresed in sodium dodecyl sulfate gels in a manner identical to that described for the native molecule. Terminal amino acid determination. Attempts were made to identify the amino-terminal amino acid by reaction of the glycoprotein (6 mg) with Dns-chloride [17]. The reaction mixture was passed through a Sephadex G-25 column. The Dnsglycoprotein derivative contained in the void volume of the column effluent was hydrolyzed in 6 M HC1 under N2 for 19 h at 105 °C. The hydrolysate was examined for Dns-amino acids by electrophoresis [17] at pH 2.0 (8 ~ formic acid in water) and at pH 4.4 (pyridine/acetic acid/water; 10:20:250, v/v). Thin-layer chromatography of the hydrolysate [18] was conducted on silica gel sheets (Eastman Kodak, Type K 301R) in three solvent systems: solvent I (chloroform/tert-amyl alcohol/acetic acid; 70:30: 3, v/v); solvent II (chloroform/tert-amyl alcohol/formic acid; 70: 30:1, v/v); solvent III (benzene/pyridine/acetic acid; 80:20:2, v/v). The fluorescent spots were detected under ultraviolet light. The COOH-terminal amino acid of glycoprotein was determined by the carboxypeptidase methods [19]. Carboxypeptidase A (20 #g, 12 units) and carboxypeptidase B (150/~g, 2 units) were reacted with 13.5 and 15.5 mg glycoprotein, respectively. Reaction tubes with appropriate controls containing enzyme but no glycoprotein were analyzed concurrently. Aliquots were removed at 0, 1 and 3 h. Free amino acids in these aliquots were quantitated using the Beckman 120C amino acid analyzer. Release of monosaccharides from porcine intimal glycoprotein by miM acid hydrolysis. The glycoprotein (12-25 mg) was dissolved in 10 ml of 0.5 M HCI and hydrolyzed at 80 °C over a 24-h period. Suitable aliquots (0.15-0.40 ml) were removed at various times. Free neutral sugars in the aliquots were quantitated by gas-liquid
29 chromatography. Hexosamine and sialic acid were determined by colorimetric procedures. Treatment of porcine intimal glycoprotein with glycosidases. The glycoprotein (1-3 mg) was incubated with neuraminidase (30 munits) in 0.25 ml of 0.1 M sodium acetate buffer, pH 4.5, at 37 °C. Aliquots (0.05 ml) were removed at various times, immersed in boiling water for 1 min and analyzed for free sialic acid. The action of fl-o-N-acetylhexosaminidase on the native glycoprotein and that exposed briefly to mild acid hydrolysis was studied as follows: Native porcine intimal glycoprotein (2-5 mg) was incubated with the enzyme (21 munits) in 1.0 ml of 50 mM sodium citrate buffer, pH 5.2, at 30 °C. Aliquots (0.2 ml) were removed and analyzed for free N-acetylhexosamine. A sample of native glycoprotein (7.6 mg) was hydrolyzed in 0.6 ml of 0.5 M HC1 at 80 °C for 2 h to remove acid-labile monosaccharides. The hydrolysate was dialyzed extensively against several changes of water and the non-dialyzable material was lyophilized. The dried protein was dissolved in 1.0 ml of 0.05 M NaOH and was acetylated by the procedure of Roseman and Daffner [20] to replace acetyl groups that may have been lost from the acetyl amino sugars during the hydrolysis. The acetylated protein was desalted by passage through a Sephadex G-25 column, the protein eluate was lyophilized and dissolved in 0.4 ml of 50 mM sodium citrate, pH 5.2. fl-o-N-Acetylhexosaminidase (20 munits) was added to this solution and the reaction mixture was incubated at 30 °C. Aliquots (0.2 ml) were taken at various times and analyzed for free N-acetylhexosamine. After 6 h of incubation, the remainder of the incubation mixture was adjusted to 2 M HCI, hydrolyzed for 16 h at 105 °C and analyzed for total hexosamine. The native and asialo-glycoproteins were treated with a-D- and fl-Dglucosidases. The various glycosidase activities present in the partially purified fl-Dglucosidase preparation were identified using o- or p-nitrophenyl glycosides. The preparation contained 15~ as much fl-D-galactosidase activity as that of fl-Dglucosidase activity. Very small amounts (less than 1 ~o of the fl-D-glucosidase activity) of fl-D-N-acetylhexosaminidase and a-o-mannosidase were present, a-DGlucosidase and a-D-galactosidase activities were not detectable. No contaminants were found in the purified a-D-glucosidase preparation. Native glycoprotein (5.9 mg) was treated with fl-D-glucosidase (2 I.U.)in 1.2 ml of 0.1 M sodium acetate buffer, pH 5.0, at 37 °C. Aliquots (0.2 ml) were removed at 0, 3 and 9 h, deionized on columns containing Dowex 50 (H +) and Dowex 1 (HCO3-), and analyzed for released neutral sugars by gas-liquid chromatography. After 9 h, a-D-glucosidase (1 I.U.) was added to the reaction mixture and the incubation continued for another 15 h. Two 0.2-ml aliquots at 12 and 24 h were analyzed for sugars. Asialo-glycoprotein (2.4 mg), obtained by treatment with neuraminidase for 24 h, was incubated with fl-o-glucosidase (2 units) in the same manner. Aliquots (0.15 ml) were taken at 0, 6 and 16 h and analyzed for neutral sugars as described above. Test for O-glycosidic linkage in the intimal glycoprotein. Two solutions of intimal glycoprotein (250/~g/ml and 1 mg/ml) were prepared in 0.5 M NaOH. The change in absorbance at 240 nm of the first solution was observed for 6 h at 25 °C and that of the other was observed for 36 h at 50 °C to detect the formation of unsaturated amino acids [21]. A similar solution containing bovine submaxillary mucin was tested to confirm the reliability of the reaction.
30 RESULTS
The molecular weight of porcine intimal glycoprotein The results of all the molecular weight determinations on the glycoprotein are summarized in Table I. Fringe displacement plots from sedimentation equilibrium analysis were linear when the glycoprotein was dissolved in NaC1 or in guanidine. HCI. Only a single protein band was visualized on polyacrylamide sodium dodecyl sulfate electrophoresis both with the native and S-carboxyamidomethylated porcine intimal glycoprotein. The variation of molecular weight as a function of acrylamide concentration was that which would be predicted for a glycoprotein containing 3 - 4 ~ carbohydrate [15]. From these data a molecular weight of 72 000 has been adopted for the intimal glycoprotein.
TABLE I MOLECULAR WEIGHT DETERMINATION OF PORCINE INTIMAL GLYCOPROTEIN Figures in parentheses indicate number of determinations. Method
Molecular weight
(A) Sedimentation equilibrium (1) NaC1, 0.1 M (2) Guanidine' HCI, 6 M
72 110 (4) 72 370 (3)
(B) Sodium dodecyl sulfate gel electrophoresis (1) Native glycoprotein 5.0 ~ acrylamide 7.5 ~ acrylamide
10.0~ acrylamide (2) S-Carboxyamidomethyl glycoprotein 5.0 ~ acrylamide 10.0~ acrylamide
85 400 (1) 77 500 (2) 74 800 (1) 75 400 (2) 70 400 (3)
NH2- and COOH-terminal amino acids Several attempts were made to identify the NHz-terminal residue of intimal glycoprotein. When the Dns-glycoprotein was hydrolyzed and the products in the hydrolysate were examined by either thin-layer chromatography or by high voltage electrophoresis, only two fluorescent spots were detected. These were identified as Dns-e-lysine and Dns-amine. In all experiments we failed to detect a Dns-a-amino acid derivative. We therefore conclude that the amino-terminal amino acid in the glycoprotein does not have an a-amino group available for reaction with Dnsreagent. Treatment of the intimal glycoproteins with carboxypeptidase B and analysis of the aliquots of the reaction mixture revealed that none of the basic amino acids was released from the glycoprotein. Several free amino acids appeared in the digest when the glycoprotein was treated with carboxypeptidase A (Fig. l). The results suggest that the COOH-terminal residue is serine. However, the data do not allow ruling out glycine as the COOH-terminal amino acid.
31 0.5
,
,
,
~
,
,
T~ °0.4 et. o >-
o
0.3
o v
./0 ~ O ' - ' / p ~ ~' (D :E
~.0 •
/ /
t~ < t~
~i// / / /
0,5
/ / / (J < c.) --
/
/
/ 0.0
2
4
' 6
..,'
I 14
I 16
I 18
I 20
I $' 2
I 24
.. ""
, 40
i 42
TIME (HOURS)
Fig. 2. Release of sialic acid from intimal glycoprotein by mild acid hydrolysis and by neuraminidase. Glycoprotein was heated in 0.5 M HC1 at 80 °C. Aliquots were analyzed for free sialic acid at 1, 4 and 6 h (Q---O); glycoprotein was treated with neuraminidase (CI. perfringens) in 0.1 M acetate, pH 4.5, at 37 °C. Aliquots were taken at 15, 24 and 42 h and analyzed for sialic acid (O---O).
z
_
3.0
Q
¢D
2.5
o
Q =E v
2.0
,=, .-1
1.5
0
F U R T H E R STUDIES ON A H I G H L Y P U R I F I E D G L Y C O P R O T E I N FROM T H E I N T I M A L R E G I O N OF PORCINE AORTA
B R U C E I. ROBERTS and P R E M A N A N D V. W A G H
Connective Tissue Laboratory, Veterans Administration Hospital, 300 E. Roosevelt Road, Little Rock, Ark. 72206 (U.S.A.) (Received December 17th, 1975)
SUMMARY
Highly purified glycoprotein from the intimal region of porcine aorta was isolated with minor modifications of the procedure described previously. The molecular weight of the glycoprotein as determined by sedimentation equilibrium method either in presence of 0.1 M NaCI or 6 M guanidine.HC1 containing fl-mercaptoethanol was 72 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native glycoprotein and its S-carboxyamidomethyl derivative at different acrylamide concentrations showed no difference in the molecular weight indicating the absence of subunits. Attempts to determine the identity of the amino-terminal acid by a dansylation technique indicated that the amino group is not flee. The carboxy-terminal amino acid was found to be serine after treatment of the glycoprotein with carboxypeptidase A. The glycoprotein did not contain an alkali-labile (O-glycosidic) carbohydrate-protein linkage as tested by the fl-elimination reaction. The release of monosaccharides from the glycoprotein as a function of time was studied employing mild acid hydrolysis (0.5 M HCI, 80 °C) and also by the use of neuraminidase, tt-Dand fl-D-glucosidases and fl-D-N-acetylglucosaminidase. From the observations on the release of monosaccharides and analogy with standard features determined by other investigators on soluble aortic glycoproteins, a prediction has been made as to the general features of the carbohydrate moiety of the glycoprotein.
INTRODUCTION
Mammalian arterial wall contains a variety of glycoproteins, some of which can be readily extracted with neutral buffers. Several soluble glycoproteins, as distinct from those which require more rigorous conditions for extraction, e.g. the use of trichloroacetic acid, high alkaline pH or urea, have been purified from bovine aorta [1, 2]. Although structural studies on the carbohydrate component of these glycoproteins have been attempted [2, 3], very little is as yet known regarding the nature of the carbohydrate moiety of glycoproteins from the aorta. We have previously reported a procedure for the isolation of a novel glycoprotein from the intimal region of porcine aorta [4]. The intimal glycoprotein contained 4 mol of hexosamine, 3 mol each of glucose and galactose, 2 mol of mannose
27 and 1 mol each of fucose and sialic acid per mol of glycoprotein (molecular weight 72 000). We report here some of the molecular properties of the glycoprotein and propose a tentative structure for the carbohydrate component consistent with the experimental results. MATERIALS AND METHODS Chemicals were obtained as follows: DEAE-cellulose (Cellex D, 0.7 mequiv./g), Coomassie brilliant blue R-250 dye and Bio-Gel P-150, Bio-Rad; Sephadex G-25 and G-15, Pharmacia Fine Chemicals; bovine pancreatic carboxypeptidase A, porcine pancreatic carboxypeptidase B, neuraminidase (Clostridium perfringens), and Dnsamino acids, Sigma Chemical Co. ; fl-o-glucosidase (sweet almond) and t~-o-glucosidase (yeast), Boehringer Mannheim Corp. All other chemicals were of reagent grade. fl-o-N-Acetylhexosaminidase (Jack bean) and bovine submaxillary mucin were gifts of Dr. Y. T. Li and Dr. Ward Pigman, respectively. Thoracic aortae from freshly killed pigs (about 6 months of age) were obtained from a local abattoir. The intima powder was prepared by the procedures previously described [4, 5]. Isolation of porcine intimal glycoprotein. The procedure for the isolation of glycoprotein described previously [4] was used with some modifications. In the present study, instead of chromatography on the first DEAE-cellulose column, a batchwise DEAE-cellulose fractionation method was used allowing us to process larger amounts of tissue. The dialyzed (NH4)2SO4 precipitate solution was added dropwise to a 1:1 (v/v) slurry of DEAE-cellulose in 0.005 M Tris.HCl buffer containing 0.001 M EDTA, pH 7.0 (standard buffer) under constant stirring. (A ratio of 2 ml packed resin per mg protein was necessary for maximal adsorption of the glycoprotein). The slurry was stirred for 3 h. The resin collected by centrifugation (1020 × g, 30 rain) was resuspended in an equal volume of standard buffer and stirred as above. After centrifugation, the supernatant fraction was discarded. Thereafter, the resin was stirred in an equal volume of standard buffer containing 0.2 M NaC1 for 3 h and centrifuged. This 0.2 M NaC1 elution was repeated two more times. The supernatant fractions from the three centrifugations were combined, dialyzed against standard buffer and lyophilized. The lyophilized product was dissolved in a small amount of standard buffer and dialyzed exhaustively against the same buffer. The solution was then applied to the second DEAE-cellulose column essentially as described previously [4]. The protein eluted in the region of the gradient corresponding to 0.16-0.18 M NaC1 contained the intimal glycoprotein. Small amounts of contaminating proteins remained due to these modifications. The contaminants were removed by passage of the glycoprotein fraction through a column of Bio-Gel P-150. Elution was performed with the standard buffer. Effluent from the column was monitored for protein at 280 nm employing an automatic ultraviolet analyzer. A major protein peak, eluting soon after the void volume of the column, was pooled, dialyzed against deionized water and lyophilized. This fraction was homogeneous as observed by disc gel electrophoresis and was identical in composition to the intimal glycoprotein reported previously [4]. Results from six different experiments were reproducible. The purity of the glycoprotein was tested by electrophoresis on polyacrytamide
28 gels. Electrophoresis was performed in 7-7.5 ~ gels using Tris/glycine buffer, pH 8.4 [6], and in 7.5~o gels using 0.1 M sodium phosphate buffer, pH 7.2, containing 0.1 sodium dodecyl sulfate [7]. Gels were stained and destained [8] using Coomassie brilliant blue R-250 dye. Analytical methods. Analysis of individual neutral sugars released from the glycoprotein by acid hydrolysis or by the action of glycosidases was accomplished by gas-liquid chromatography of the alditol acetates [9, 10]. Sialic acid was determined by the thiobarbituric acid method [11]. Hexosamine was quantitated by the procedure of Gatt and Berman [12]. Glucosamine was identified as the sole amino sugar by use of a Beckman 120C amino acid analyzer following hydrolysis of the glycoprotein in 2 M HCI for 16 h at 100 °C. The method of Reissig et al. [13] was used for N-acetyl amino sugar analysis. Other analytical procedures have been previously described [4]. Molecular weight determhration. The molecular weight of porcine intimal glycoprotein was determined both by sedimentation equilibrium method [14] using a Beckman Model E ultracentrifuge and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [7, 15]. In sedimentation equilibrium studies, the glycoprotein was dissolved in 0.1 M NaC1 and in 6 M guanidine.HCl containing 0.1~o flmercaptoethanol. Mixtures containing porcine intimal glycoprotein and standard proteins of known molecular weights were electrophoresed on 5, 7.5 and 10~ polyacrylamide gels in 0.1 M sodium phosphate buffer at pH 7.2 containing 1 ~ sodium dodecyi sulfate. Electrophoresis was performed at a current of 3.3-4 mA (25 oC) per tube for 1.5-5 h. In a separate experiment, the glycoprotein was first reduced with/3mercaptoethanol in 6 M urea and then alkylated with iodoacetamide [16]. The Scarboxyarnidomethylated derivative was electrophoresed in sodium dodecyl sulfate gels in a manner identical to that described for the native molecule. Terminal amino acid determination. Attempts were made to identify the amino-terminal amino acid by reaction of the glycoprotein (6 mg) with Dns-chloride [17]. The reaction mixture was passed through a Sephadex G-25 column. The Dnsglycoprotein derivative contained in the void volume of the column effluent was hydrolyzed in 6 M HC1 under N2 for 19 h at 105 °C. The hydrolysate was examined for Dns-amino acids by electrophoresis [17] at pH 2.0 (8 ~ formic acid in water) and at pH 4.4 (pyridine/acetic acid/water; 10:20:250, v/v). Thin-layer chromatography of the hydrolysate [18] was conducted on silica gel sheets (Eastman Kodak, Type K 301R) in three solvent systems: solvent I (chloroform/tert-amyl alcohol/acetic acid; 70:30: 3, v/v); solvent II (chloroform/tert-amyl alcohol/formic acid; 70: 30:1, v/v); solvent III (benzene/pyridine/acetic acid; 80:20:2, v/v). The fluorescent spots were detected under ultraviolet light. The COOH-terminal amino acid of glycoprotein was determined by the carboxypeptidase methods [19]. Carboxypeptidase A (20 #g, 12 units) and carboxypeptidase B (150/~g, 2 units) were reacted with 13.5 and 15.5 mg glycoprotein, respectively. Reaction tubes with appropriate controls containing enzyme but no glycoprotein were analyzed concurrently. Aliquots were removed at 0, 1 and 3 h. Free amino acids in these aliquots were quantitated using the Beckman 120C amino acid analyzer. Release of monosaccharides from porcine intimal glycoprotein by miM acid hydrolysis. The glycoprotein (12-25 mg) was dissolved in 10 ml of 0.5 M HCI and hydrolyzed at 80 °C over a 24-h period. Suitable aliquots (0.15-0.40 ml) were removed at various times. Free neutral sugars in the aliquots were quantitated by gas-liquid
29 chromatography. Hexosamine and sialic acid were determined by colorimetric procedures. Treatment of porcine intimal glycoprotein with glycosidases. The glycoprotein (1-3 mg) was incubated with neuraminidase (30 munits) in 0.25 ml of 0.1 M sodium acetate buffer, pH 4.5, at 37 °C. Aliquots (0.05 ml) were removed at various times, immersed in boiling water for 1 min and analyzed for free sialic acid. The action of fl-o-N-acetylhexosaminidase on the native glycoprotein and that exposed briefly to mild acid hydrolysis was studied as follows: Native porcine intimal glycoprotein (2-5 mg) was incubated with the enzyme (21 munits) in 1.0 ml of 50 mM sodium citrate buffer, pH 5.2, at 30 °C. Aliquots (0.2 ml) were removed and analyzed for free N-acetylhexosamine. A sample of native glycoprotein (7.6 mg) was hydrolyzed in 0.6 ml of 0.5 M HC1 at 80 °C for 2 h to remove acid-labile monosaccharides. The hydrolysate was dialyzed extensively against several changes of water and the non-dialyzable material was lyophilized. The dried protein was dissolved in 1.0 ml of 0.05 M NaOH and was acetylated by the procedure of Roseman and Daffner [20] to replace acetyl groups that may have been lost from the acetyl amino sugars during the hydrolysis. The acetylated protein was desalted by passage through a Sephadex G-25 column, the protein eluate was lyophilized and dissolved in 0.4 ml of 50 mM sodium citrate, pH 5.2. fl-o-N-Acetylhexosaminidase (20 munits) was added to this solution and the reaction mixture was incubated at 30 °C. Aliquots (0.2 ml) were taken at various times and analyzed for free N-acetylhexosamine. After 6 h of incubation, the remainder of the incubation mixture was adjusted to 2 M HCI, hydrolyzed for 16 h at 105 °C and analyzed for total hexosamine. The native and asialo-glycoproteins were treated with a-D- and fl-Dglucosidases. The various glycosidase activities present in the partially purified fl-Dglucosidase preparation were identified using o- or p-nitrophenyl glycosides. The preparation contained 15~ as much fl-D-galactosidase activity as that of fl-Dglucosidase activity. Very small amounts (less than 1 ~o of the fl-D-glucosidase activity) of fl-D-N-acetylhexosaminidase and a-o-mannosidase were present, a-DGlucosidase and a-D-galactosidase activities were not detectable. No contaminants were found in the purified a-D-glucosidase preparation. Native glycoprotein (5.9 mg) was treated with fl-D-glucosidase (2 I.U.)in 1.2 ml of 0.1 M sodium acetate buffer, pH 5.0, at 37 °C. Aliquots (0.2 ml) were removed at 0, 3 and 9 h, deionized on columns containing Dowex 50 (H +) and Dowex 1 (HCO3-), and analyzed for released neutral sugars by gas-liquid chromatography. After 9 h, a-D-glucosidase (1 I.U.) was added to the reaction mixture and the incubation continued for another 15 h. Two 0.2-ml aliquots at 12 and 24 h were analyzed for sugars. Asialo-glycoprotein (2.4 mg), obtained by treatment with neuraminidase for 24 h, was incubated with fl-o-glucosidase (2 units) in the same manner. Aliquots (0.15 ml) were taken at 0, 6 and 16 h and analyzed for neutral sugars as described above. Test for O-glycosidic linkage in the intimal glycoprotein. Two solutions of intimal glycoprotein (250/~g/ml and 1 mg/ml) were prepared in 0.5 M NaOH. The change in absorbance at 240 nm of the first solution was observed for 6 h at 25 °C and that of the other was observed for 36 h at 50 °C to detect the formation of unsaturated amino acids [21]. A similar solution containing bovine submaxillary mucin was tested to confirm the reliability of the reaction.
30 RESULTS
The molecular weight of porcine intimal glycoprotein The results of all the molecular weight determinations on the glycoprotein are summarized in Table I. Fringe displacement plots from sedimentation equilibrium analysis were linear when the glycoprotein was dissolved in NaC1 or in guanidine. HCI. Only a single protein band was visualized on polyacrylamide sodium dodecyl sulfate electrophoresis both with the native and S-carboxyamidomethylated porcine intimal glycoprotein. The variation of molecular weight as a function of acrylamide concentration was that which would be predicted for a glycoprotein containing 3 - 4 ~ carbohydrate [15]. From these data a molecular weight of 72 000 has been adopted for the intimal glycoprotein.
TABLE I MOLECULAR WEIGHT DETERMINATION OF PORCINE INTIMAL GLYCOPROTEIN Figures in parentheses indicate number of determinations. Method
Molecular weight
(A) Sedimentation equilibrium (1) NaC1, 0.1 M (2) Guanidine' HCI, 6 M
72 110 (4) 72 370 (3)
(B) Sodium dodecyl sulfate gel electrophoresis (1) Native glycoprotein 5.0 ~ acrylamide 7.5 ~ acrylamide
10.0~ acrylamide (2) S-Carboxyamidomethyl glycoprotein 5.0 ~ acrylamide 10.0~ acrylamide
85 400 (1) 77 500 (2) 74 800 (1) 75 400 (2) 70 400 (3)
NH2- and COOH-terminal amino acids Several attempts were made to identify the NHz-terminal residue of intimal glycoprotein. When the Dns-glycoprotein was hydrolyzed and the products in the hydrolysate were examined by either thin-layer chromatography or by high voltage electrophoresis, only two fluorescent spots were detected. These were identified as Dns-e-lysine and Dns-amine. In all experiments we failed to detect a Dns-a-amino acid derivative. We therefore conclude that the amino-terminal amino acid in the glycoprotein does not have an a-amino group available for reaction with Dnsreagent. Treatment of the intimal glycoproteins with carboxypeptidase B and analysis of the aliquots of the reaction mixture revealed that none of the basic amino acids was released from the glycoprotein. Several free amino acids appeared in the digest when the glycoprotein was treated with carboxypeptidase A (Fig. l). The results suggest that the COOH-terminal residue is serine. However, the data do not allow ruling out glycine as the COOH-terminal amino acid.
31 0.5
,
,
,
~
,
,
T~ °0.4 et. o >-
o
0.3
o v
./0 ~ O ' - ' / p ~ ~' (D :E
~.0 •
/ /
t~ < t~
~i// / / /
0,5
/ / / (J < c.) --
/
/
/ 0.0
2
4
' 6
..,'
I 14
I 16
I 18
I 20
I $' 2
I 24
.. ""
, 40
i 42
TIME (HOURS)
Fig. 2. Release of sialic acid from intimal glycoprotein by mild acid hydrolysis and by neuraminidase. Glycoprotein was heated in 0.5 M HC1 at 80 °C. Aliquots were analyzed for free sialic acid at 1, 4 and 6 h (Q---O); glycoprotein was treated with neuraminidase (CI. perfringens) in 0.1 M acetate, pH 4.5, at 37 °C. Aliquots were taken at 15, 24 and 42 h and analyzed for sialic acid (O---O).
z
_
3.0
Q
¢D
2.5
o
Q =E v
2.0
,=, .-1
1.5
0
