Biochimica et Biophysica Acta, 427 (1976) 91-106
© Elsevier ScientificPublishing Company,Amsterdam- Printed in The Netherlands BBA 37285 STRUCTURAL STUDIES ON A GLYCOPROTEIN ISOLATED FROM ALVEOLI OF PATIENTS WITH ALVEOLAR PROTEINOSIS
S. N. BHATTACHARYYA,S. SAHU and W. S. LYNN Departments of Biochemistry and Medicine, Duke University Medical Center, Durham, N.C. 27710 (U.S.A.)
(Received July 22nd, 1975)
SUMMARY A major glycoprotein (36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 tool of mannose, 4.0 tool of galactose, 6.0 tool of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NHzterminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4 ~ hydroxyproline, 14 ~o proline, 28 ~ glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of-Gly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of aspartic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.
92 INTRODUCTION Alveolar proteinosis [1] is a chronic pulmonary disease of unknown pathogenesis in which the alveoli and terminal bronchioles of the lung are filled with periodic acid-Schiff-positive amorphous material. This material, which is insoluble in isotonic saline, can be removed by pulmonary lavage [2, 3]. Analyses [4] by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of three major proteins, the molecular weights of which are 69 000, 62 000 and 36 000, respectively. The 62 000 and 36 000 molecular weight proteins were glycoproteins and found to contain sialic acid, galactose, mannose, fucose, glucosamine, hydroxyproline and relatively high amounts of glycine. The same two glycoproteins with similar compositions were found in lavage material and lamellar bodies isolated from normal animal lungs [5]. The 36 000 molecular weight glycoprotein was the major one in all cases. The presence of hydroxyproline (5 residues) and a relatively high amount of glycine (50 residues) in the 36 000 molecular weight glycoprotein suggests that short collagen-like regions may be present in the glycoprotein. End group analysis and extensive attempts to purify this protein all indicated the presence of only one peptide. This glycoprotein contains three residues of methionine and treatment with cyanogen bromide (CNBr) [6] should therefore yield four fragments. The expected four peptides were obtained in good yield after treatment of the 36 000 molecular weight glycoprotein with CNBr. Their molecular weights, chemical compositions and Nterminal amino acids are presented. A partial amino-terminal sequence of the 18 000 molecular weight CNBr peptide is also presented. In addition, studies on the mode of attachment of the carbohydrate moieties to the peptide chain are reported. MATERIALS AND METHODS All chemicals are of reagent grade. Acrylamide, bisacrylamide, ammonium persulfate, N,N,N',N'-tetramethylethylene diamine (TEMED), and different Bio-Gels for column preparation were all purchased from Bio-Rad Laboratories, Richmond, Calif., U.S.A. Pure sodium dodecyl sulfate was obtained from B.D.H. Chemicals Ltd., Poole, England. Cyanogen bromide, dansyl chloride (10 ~ w/v, in acetone), phenylisothiocyanate and trifluoroacetic acid (both sequanal grade) were purchased from Pierce Chemical Company, Rockford, Ill., U.S.A. Pronase (Grade B) was purchased from Calbiochem, San Diego, Calif., U.S.A. Cheng-Chin polyamide layer sheets were obtained from Gallard-Schlesinger, Carle Place, N.Y., U.S.A.
Isolation of lavage material Lavage material was obtained from lungs of patients with alveolar proteinosis as described [4]. The lavage material was suspended in cold isotonic saline and was centrifuged in cold at 250 × g for 10 rain to remove alveolar macrophages and other cells. The supernate was then centrifuged in the cold at 25 000 × g for 15 rain. The precipitate, in particulate form, obtained by this centrifugegation was suspended in 5 mM Tris.HC1 buffer, containing 0.25 M sucrose and 1 mM EDTA (pH 7.5), and homogenized. To remove completely any remaining cells and alveolar organelles, the
93 homogenized material was layered on 30 ~ sucrose (by wt.) and centrifuged at 4 °C at 75 000 × g for 1 h. The material at the interface was removed, suspended in 5 mM Tris.HC1 buffer containing 1 mM EDTA (pH 7.5), and centrifuged at 100 000 × g for 30 min. The precipitate was collected and washed five times with the same buffer. The precipitate was lyophilized and stored at --20 °C. No cells or cellular organelles were present, as observed by electron microscopy.
Isolation of glycoprotein The lyophilized material was delipidated by five extractions with chloroform/ methanol (2:1, v/v). The delipidated lavage precipitate was dissolved in 40 mM Tris.HC1 buffer (pH 7.5) containing 0.1 ~ sodium dodecyl sulfate and 0.05~o 2mercaptoethanol. Complete solubilization of this material could be achieved only in the presence of thiol reagents. Any insoluble material formed was removed by centrifugation. The solubilized precipitate was applied to a column (2.5 × 80 cm) of Bio-Gel P-200 equilibrated with 20 mM Tris. HC1 buffer (pH 7.5) containing 0.1 ~ sodium dodecyl sulfate, 0.05 ~o 2-mercaptoethanol and 0.2 ~ sodium azide and eluted with the equilibrating buffer. Three peaks were observed as determined by absorbance measurements at 280 nm. The third peak contained no peptides other than the 36 000 molecular weight glycoprotein and sodium dodecyl sulfate-acrylamide gel electrophoresis gave a single band. The fractions from Bio-Gel P-200 column containing only the 36 000 molecular glycoprotein were combined and dialyzed against 20 mM Tris-HC1 buffer (pH 7.5) for 24 h at 4 °C to remove mercaptoethanol and minimize the content of sodium dodecyl sulfate. The dialyzate was then lyophilized.
Chemical analyses and sodium dodecyl sulfate-polyacrylamide gel electrophoresis Total protein was estimated by the method of Lowry et al. [7], using bovine serum albumin as the standard. Total carbohydrate was determined by the phenolsulfuric acid method [8], using glucose as standard. Sialic acid was estimated by the thiobarbituric acid assay method [9], after hydrolyzing the glycopeptides in 0.1 M H2SO4 at 80 °C for 1 h. Sodium dodecyl sulfate-acrylamide gel electrophoresis was performed by the method of Weber and Osborn [10]. The gels were stained with either Coomassie blue for peptides or periodic acid-Schiff reagent for glycopeptides [11 ]. Molecular weight estimation for peptides were made according to the method of Weber and Osborn [10] using bovine serum albumin, ovalbumin, concanavalin A, cytochrome c, and insulin as standards.
CNBr treatment of the 36 000 molecular weight glycoprotein The lyophilized glycoprotein from Bio-Gel P-200 column (10 mg of protein) was treated with CNBr in 70 ~ formic acid according to the method of Givol and Porter [12]. The solution was kept at room temperature under nitrogen for 24 h and then centrifuged to remove a small precipitate formed during the reaction. The supernatant was diluted with water and lyophilized. The lyophilized material was dissolved in 10 mM Tris.HCI buffer (pH 7.5) containing 0 . 0 5 ~ sodium dodecyl sulfate, 0.01 ~ 2-mercaptoethanol and 0.02~o
94 sodium azide and applied to a column (2.5 × 65 cm) of Bio-Gel P-60 equilibrated with the same buffer. The column was eluted with the equilibrating buffer. The fractions from Bio-Gel P-60 column containing the lowest peptide ( ~ 1,000 molecular weight) were combined and passed through Bio-Gel P-2 (2.5 × 30 cm) column equilibrated with 10 mM Tris. HC1 buffer (pH 7.5) containing 0.05 ~ sodium dodecyl sulfate, 0.01 ~ 2-mercaptoethanol and 0.02 ~ sodium azide. The peptide was eluted with the equilibrating buffer. Absorbance measurement at 280 nm was used to detect the eluted material. Amino acid analyses were performed by the method of Spackman [13] on a Beckman 120 B Analyzer. The peptides were hydrolyzed in 6 M HC1 in an evacuated sealed tube and in the presence of nitrogen at 110 °C for 24, 48 or 72 h. Amino acid analyses were done on the peptides isolated by Bio-Gel column chromatography, as well as on the peptides eluted from sodium dodecyl sulfate-acrylamide gels according to the method of Weiner et al. [14]. Hydroxylysine and hydroxyproline analyses were done by the method of Piez and Morris [15] and also using a single column (Durrum DC-1A) and a temperature gradient. The first 30 min of elution of amino acids was at 30 °C. The temperature was then increased (over the next 15 min) to 60 °C. Hydroxyamino acids were well separated in this column. Cysteine was determined as cysteic acid as described by Moore [16]. Before amino acid analyses, the acid hydrolysates (after removal of acid) of CNBr peptides were treated with pyridine acetate buffer (pH 6.5) at 105 °C for 1-2 h for the conversion of homoserine lactone to homoserine, as described by Ambler [17]. Therefore, the reported values for homoserine were the sum of the values for homoserine and homoserine lactone. Hexosamines were also estimated on a single column of amino acid analyzer after hydrolyzing the peptides in an evacuated sealed tube in 4 M HC1 for 6 h at 110 °C, as described by Spiro [18]. Under this condition, the recovery of hexosamines was 85 ~ . The neutral sugars were isolated through coupled columns of Dowex 50-X4, 200-400 mesh (H +) and Dowex l-X8, 200--400 mesh (formate) after hydrolyzing the peptides in an evacuated sealed tube with 1 M HC1 for 5 h at 110 °C, as described by Spiro [18]. The loss in the amount of neutral sugars by this method was within 15 ~ . The water effluents (water wash equal 4-5 column volumes) from the coupled columns were combined and lyophilized. The neutral sugars in this material were estimated by automated borate-complex anion exchange chromatography [18]. Fucose was also determined by Dische-Shettles cysteine-sulfuric acid reaction, as described by Spiro [l 9], after hydrolyzing the peptides in an evacuated sealed tube with 0.1 MH2 SO4 at 100 °C for 8 h. Not more than 12 ~ of the fucose was destroyed under this condition.
End-group analyses Qualitative identification of NH/-terminal amino acid residues on the peptides isolated from Bio-Gel column was performed by the dansyl procedure as described by Gray [20] with the modifications suggested by Hartley [21]. The NH2-terminal amino acid residues on the peptides eluted from sodium dodecyl sulfate-acrylamide gels were also determined by the method of Weiner et al. [14]. In both cases, similar NHz-terminal residue was obtained with respect to each peptide. The COOH-terminal residue was not identified.
95
Proteolytic digestion and gel filtration of the pronase digest of the glycoprotein About 50 mg of the 36 000 molecular weight glycoprotein was digested with pronase in 0.15 M Tris acetate buffer, p H 7.8, containing 0.015 M calcium acetate, for 48 h at 45 °C in the presence of toluene. Pronase was added initially to equal 10 of the glycoprotein weight, with further addition of 10 ~ of the substrate weight being made at 24 h. The pH of the reaction mixture was readjusted to 7.8 at 24 h with 1 M Tris. At the end of pronase digestion, the solution was lyophilized and the residue was dissolved in 0.1 M pyridine acetate buffer at pH 5.0 and fractionated on Bio-Gel P-4 column (2.5 x 80 cm) equilibrated with the same buffer as above. Elution was achieved with the 0,1 M pyridine acetate buffer at p H 5.0 at a rate of 12 ml per hour. Fractions of 2 ml were collected and aliquots of these were analyzed by phenol-sulfuric acid [8] and ninhydrin [22] reactions. The fractions (Peak I, Fig. 5) containing carbohydrate were combined and lyophilized. The protein concentration in these fractions represented about 4 ~ of the starting material.
Chromatography of glycopeptides (Bio-Gel P-4 column fraction) on Dowex 50-X2 The lyophilized material containing carbohydrate from Bio-Gel P-4 column was dissolved in 0.1 M formic acid and titrated to pH 5.0 with sodium hydroxide. The solution was then passed through a small column (1 × 5 cm) of Dowex 50-X2, 200-400 mesh (H +) (previously washed with 2 M NaOH, water, 3 M HC1 and water, in that order). After applying the sample to the column, the column was washed with 3-4 column volumes of cold deionized water and then with cold 1.5 M NH4OH. The acidic fraction (Fraction I) and basic fraction (Fraction II) were combined separately and lyophilized. The basic fraction, which was not soluble in water, was dissolved in the solvent system for paper electrophoresis.
Carbohydrate linkage in the 36 000 molecular weight glycoprotein The reductive cleavage of the native glycoprotein or glycopeptide fraction I o f the pronase digested peptide by alkaline borohydride in different periods of time was carried out as described by Tanaka et al. [23]. N-acetylglucosamine-aspartic acid complex from the acidic glycopeptide fraction (Fraction I) was isolated according to the procedure of Marshall and Neuberger [24]. The glycopeptide fraction was hydrolyzed in 2 M HC1 at 100 °C for 15 min. After cooling, the solution was passed through Amberlite CG-4B (2 × 15 cm) column in the O H - form. The column was eluted with deionized water (150 ml) and the p H of the aqueous effluent was adjusted to 5.6 with 3 M HCI. The solution was lyophilized. The residue was taken up in 1 ml of water and placed in Dowex 50-X4 (200-400 mesh) in the H ÷ form (1 x 4 cm) together with 2 x 1 ml water washings. After washing the column with 80 ml of water, elution was carried out with aqueous HC1 gradient (100 ml water and 100 ml 2 M HC1). Each 3 ml fraction was analyzed for ninhydrin-positive [24] material (Fig. 6). Paper chromatography for isolating N-acetylglucosamine-aspartic acid complex was carried out on Whatman No. 1 paper by the descending technique and the solvent system used was butanol/acetic acid/water (4:1:5) as described by Spiro [18]. The paper was stained with ninhydrin reagent as described by Marshall and Neuberger [24]. The individual components on the paper were located with the help of
96 ninhydrin-stained guide strips and eluted with 0.1 M acetic acid. After lyophilization, the residues were hydrolyzed for 4 h in an evacuated sealed tube in 4 M HC1 at 105 °C for the determination of amino acids and hexosamines [24].
High voltage paper electrophoresis High voltage paper electrophoresis (CAMAC HVE System, U.S.A.) of the pronase-digested peptides (Fraction II) of the 36 000 molecular weight glycoprotein was carried out at 3500 V (70 mA) for 45 min. The solvent system used was acetic acid/formic acid/water (10:5:8). The peptides were dissolved in the same solvent system and spotted on the paper. The peptides which moved toward the cathode were located by ninhydrin reagent and with the help of ninhydrin-stained guide strip, the peptides were eluted from paper with 0.1 M acetic acid. The solution was lyophilized. The residues were hydrolyzed in 6 M HC1 in an evacuated sealed tube under nitrogen at 110 °C for 24 h for the analysis of amino acid.
Amino-terminal sequence analysis of the 18 000 molecular weight CNBr peptide Partial amino-terminal sequence analysis on the 18 000 molecular weight CNBr peptide was done manually according to the sodium dodecyl sulfate-dansylEdman degradation method as described by Weiner et al. [14]. RESULTS The intact glycoprotein was separated from the lavage material by Bio-Gel P-200 column chromatography, as seen in Fig. 1. The third peak contained the purified glycoprotein. The second peak contained the 69 000 and 62 000 molecular weight proteins, as described before [4, 5]. The purified glycoprotein showed a single band on sodium dodecyl sulfate-acrylamide gel electrophoresis (Fig. 4A) with a mobility corresponding to molecular weight 36 000. The molecular weight estimation of the native glycoprotein on sodium dodecyl
36 000
0.5
=E 0.4 69000 + 62000
~Z 0.3 .,
© Elsevier ScientificPublishing Company,Amsterdam- Printed in The Netherlands BBA 37285 STRUCTURAL STUDIES ON A GLYCOPROTEIN ISOLATED FROM ALVEOLI OF PATIENTS WITH ALVEOLAR PROTEINOSIS
S. N. BHATTACHARYYA,S. SAHU and W. S. LYNN Departments of Biochemistry and Medicine, Duke University Medical Center, Durham, N.C. 27710 (U.S.A.)
(Received July 22nd, 1975)
SUMMARY A major glycoprotein (36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 tool of mannose, 4.0 tool of galactose, 6.0 tool of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NHzterminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4 ~ hydroxyproline, 14 ~o proline, 28 ~ glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of-Gly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of aspartic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.
92 INTRODUCTION Alveolar proteinosis [1] is a chronic pulmonary disease of unknown pathogenesis in which the alveoli and terminal bronchioles of the lung are filled with periodic acid-Schiff-positive amorphous material. This material, which is insoluble in isotonic saline, can be removed by pulmonary lavage [2, 3]. Analyses [4] by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of three major proteins, the molecular weights of which are 69 000, 62 000 and 36 000, respectively. The 62 000 and 36 000 molecular weight proteins were glycoproteins and found to contain sialic acid, galactose, mannose, fucose, glucosamine, hydroxyproline and relatively high amounts of glycine. The same two glycoproteins with similar compositions were found in lavage material and lamellar bodies isolated from normal animal lungs [5]. The 36 000 molecular weight glycoprotein was the major one in all cases. The presence of hydroxyproline (5 residues) and a relatively high amount of glycine (50 residues) in the 36 000 molecular weight glycoprotein suggests that short collagen-like regions may be present in the glycoprotein. End group analysis and extensive attempts to purify this protein all indicated the presence of only one peptide. This glycoprotein contains three residues of methionine and treatment with cyanogen bromide (CNBr) [6] should therefore yield four fragments. The expected four peptides were obtained in good yield after treatment of the 36 000 molecular weight glycoprotein with CNBr. Their molecular weights, chemical compositions and Nterminal amino acids are presented. A partial amino-terminal sequence of the 18 000 molecular weight CNBr peptide is also presented. In addition, studies on the mode of attachment of the carbohydrate moieties to the peptide chain are reported. MATERIALS AND METHODS All chemicals are of reagent grade. Acrylamide, bisacrylamide, ammonium persulfate, N,N,N',N'-tetramethylethylene diamine (TEMED), and different Bio-Gels for column preparation were all purchased from Bio-Rad Laboratories, Richmond, Calif., U.S.A. Pure sodium dodecyl sulfate was obtained from B.D.H. Chemicals Ltd., Poole, England. Cyanogen bromide, dansyl chloride (10 ~ w/v, in acetone), phenylisothiocyanate and trifluoroacetic acid (both sequanal grade) were purchased from Pierce Chemical Company, Rockford, Ill., U.S.A. Pronase (Grade B) was purchased from Calbiochem, San Diego, Calif., U.S.A. Cheng-Chin polyamide layer sheets were obtained from Gallard-Schlesinger, Carle Place, N.Y., U.S.A.
Isolation of lavage material Lavage material was obtained from lungs of patients with alveolar proteinosis as described [4]. The lavage material was suspended in cold isotonic saline and was centrifuged in cold at 250 × g for 10 rain to remove alveolar macrophages and other cells. The supernate was then centrifuged in the cold at 25 000 × g for 15 rain. The precipitate, in particulate form, obtained by this centrifugegation was suspended in 5 mM Tris.HC1 buffer, containing 0.25 M sucrose and 1 mM EDTA (pH 7.5), and homogenized. To remove completely any remaining cells and alveolar organelles, the
93 homogenized material was layered on 30 ~ sucrose (by wt.) and centrifuged at 4 °C at 75 000 × g for 1 h. The material at the interface was removed, suspended in 5 mM Tris.HC1 buffer containing 1 mM EDTA (pH 7.5), and centrifuged at 100 000 × g for 30 min. The precipitate was collected and washed five times with the same buffer. The precipitate was lyophilized and stored at --20 °C. No cells or cellular organelles were present, as observed by electron microscopy.
Isolation of glycoprotein The lyophilized material was delipidated by five extractions with chloroform/ methanol (2:1, v/v). The delipidated lavage precipitate was dissolved in 40 mM Tris.HC1 buffer (pH 7.5) containing 0.1 ~ sodium dodecyl sulfate and 0.05~o 2mercaptoethanol. Complete solubilization of this material could be achieved only in the presence of thiol reagents. Any insoluble material formed was removed by centrifugation. The solubilized precipitate was applied to a column (2.5 × 80 cm) of Bio-Gel P-200 equilibrated with 20 mM Tris. HC1 buffer (pH 7.5) containing 0.1 ~ sodium dodecyl sulfate, 0.05 ~o 2-mercaptoethanol and 0.2 ~ sodium azide and eluted with the equilibrating buffer. Three peaks were observed as determined by absorbance measurements at 280 nm. The third peak contained no peptides other than the 36 000 molecular weight glycoprotein and sodium dodecyl sulfate-acrylamide gel electrophoresis gave a single band. The fractions from Bio-Gel P-200 column containing only the 36 000 molecular glycoprotein were combined and dialyzed against 20 mM Tris-HC1 buffer (pH 7.5) for 24 h at 4 °C to remove mercaptoethanol and minimize the content of sodium dodecyl sulfate. The dialyzate was then lyophilized.
Chemical analyses and sodium dodecyl sulfate-polyacrylamide gel electrophoresis Total protein was estimated by the method of Lowry et al. [7], using bovine serum albumin as the standard. Total carbohydrate was determined by the phenolsulfuric acid method [8], using glucose as standard. Sialic acid was estimated by the thiobarbituric acid assay method [9], after hydrolyzing the glycopeptides in 0.1 M H2SO4 at 80 °C for 1 h. Sodium dodecyl sulfate-acrylamide gel electrophoresis was performed by the method of Weber and Osborn [10]. The gels were stained with either Coomassie blue for peptides or periodic acid-Schiff reagent for glycopeptides [11 ]. Molecular weight estimation for peptides were made according to the method of Weber and Osborn [10] using bovine serum albumin, ovalbumin, concanavalin A, cytochrome c, and insulin as standards.
CNBr treatment of the 36 000 molecular weight glycoprotein The lyophilized glycoprotein from Bio-Gel P-200 column (10 mg of protein) was treated with CNBr in 70 ~ formic acid according to the method of Givol and Porter [12]. The solution was kept at room temperature under nitrogen for 24 h and then centrifuged to remove a small precipitate formed during the reaction. The supernatant was diluted with water and lyophilized. The lyophilized material was dissolved in 10 mM Tris.HCI buffer (pH 7.5) containing 0 . 0 5 ~ sodium dodecyl sulfate, 0.01 ~ 2-mercaptoethanol and 0.02~o
94 sodium azide and applied to a column (2.5 × 65 cm) of Bio-Gel P-60 equilibrated with the same buffer. The column was eluted with the equilibrating buffer. The fractions from Bio-Gel P-60 column containing the lowest peptide ( ~ 1,000 molecular weight) were combined and passed through Bio-Gel P-2 (2.5 × 30 cm) column equilibrated with 10 mM Tris. HC1 buffer (pH 7.5) containing 0.05 ~ sodium dodecyl sulfate, 0.01 ~ 2-mercaptoethanol and 0.02 ~ sodium azide. The peptide was eluted with the equilibrating buffer. Absorbance measurement at 280 nm was used to detect the eluted material. Amino acid analyses were performed by the method of Spackman [13] on a Beckman 120 B Analyzer. The peptides were hydrolyzed in 6 M HC1 in an evacuated sealed tube and in the presence of nitrogen at 110 °C for 24, 48 or 72 h. Amino acid analyses were done on the peptides isolated by Bio-Gel column chromatography, as well as on the peptides eluted from sodium dodecyl sulfate-acrylamide gels according to the method of Weiner et al. [14]. Hydroxylysine and hydroxyproline analyses were done by the method of Piez and Morris [15] and also using a single column (Durrum DC-1A) and a temperature gradient. The first 30 min of elution of amino acids was at 30 °C. The temperature was then increased (over the next 15 min) to 60 °C. Hydroxyamino acids were well separated in this column. Cysteine was determined as cysteic acid as described by Moore [16]. Before amino acid analyses, the acid hydrolysates (after removal of acid) of CNBr peptides were treated with pyridine acetate buffer (pH 6.5) at 105 °C for 1-2 h for the conversion of homoserine lactone to homoserine, as described by Ambler [17]. Therefore, the reported values for homoserine were the sum of the values for homoserine and homoserine lactone. Hexosamines were also estimated on a single column of amino acid analyzer after hydrolyzing the peptides in an evacuated sealed tube in 4 M HC1 for 6 h at 110 °C, as described by Spiro [18]. Under this condition, the recovery of hexosamines was 85 ~ . The neutral sugars were isolated through coupled columns of Dowex 50-X4, 200-400 mesh (H +) and Dowex l-X8, 200--400 mesh (formate) after hydrolyzing the peptides in an evacuated sealed tube with 1 M HC1 for 5 h at 110 °C, as described by Spiro [18]. The loss in the amount of neutral sugars by this method was within 15 ~ . The water effluents (water wash equal 4-5 column volumes) from the coupled columns were combined and lyophilized. The neutral sugars in this material were estimated by automated borate-complex anion exchange chromatography [18]. Fucose was also determined by Dische-Shettles cysteine-sulfuric acid reaction, as described by Spiro [l 9], after hydrolyzing the peptides in an evacuated sealed tube with 0.1 MH2 SO4 at 100 °C for 8 h. Not more than 12 ~ of the fucose was destroyed under this condition.
End-group analyses Qualitative identification of NH/-terminal amino acid residues on the peptides isolated from Bio-Gel column was performed by the dansyl procedure as described by Gray [20] with the modifications suggested by Hartley [21]. The NH2-terminal amino acid residues on the peptides eluted from sodium dodecyl sulfate-acrylamide gels were also determined by the method of Weiner et al. [14]. In both cases, similar NHz-terminal residue was obtained with respect to each peptide. The COOH-terminal residue was not identified.
95
Proteolytic digestion and gel filtration of the pronase digest of the glycoprotein About 50 mg of the 36 000 molecular weight glycoprotein was digested with pronase in 0.15 M Tris acetate buffer, p H 7.8, containing 0.015 M calcium acetate, for 48 h at 45 °C in the presence of toluene. Pronase was added initially to equal 10 of the glycoprotein weight, with further addition of 10 ~ of the substrate weight being made at 24 h. The pH of the reaction mixture was readjusted to 7.8 at 24 h with 1 M Tris. At the end of pronase digestion, the solution was lyophilized and the residue was dissolved in 0.1 M pyridine acetate buffer at pH 5.0 and fractionated on Bio-Gel P-4 column (2.5 x 80 cm) equilibrated with the same buffer as above. Elution was achieved with the 0,1 M pyridine acetate buffer at p H 5.0 at a rate of 12 ml per hour. Fractions of 2 ml were collected and aliquots of these were analyzed by phenol-sulfuric acid [8] and ninhydrin [22] reactions. The fractions (Peak I, Fig. 5) containing carbohydrate were combined and lyophilized. The protein concentration in these fractions represented about 4 ~ of the starting material.
Chromatography of glycopeptides (Bio-Gel P-4 column fraction) on Dowex 50-X2 The lyophilized material containing carbohydrate from Bio-Gel P-4 column was dissolved in 0.1 M formic acid and titrated to pH 5.0 with sodium hydroxide. The solution was then passed through a small column (1 × 5 cm) of Dowex 50-X2, 200-400 mesh (H +) (previously washed with 2 M NaOH, water, 3 M HC1 and water, in that order). After applying the sample to the column, the column was washed with 3-4 column volumes of cold deionized water and then with cold 1.5 M NH4OH. The acidic fraction (Fraction I) and basic fraction (Fraction II) were combined separately and lyophilized. The basic fraction, which was not soluble in water, was dissolved in the solvent system for paper electrophoresis.
Carbohydrate linkage in the 36 000 molecular weight glycoprotein The reductive cleavage of the native glycoprotein or glycopeptide fraction I o f the pronase digested peptide by alkaline borohydride in different periods of time was carried out as described by Tanaka et al. [23]. N-acetylglucosamine-aspartic acid complex from the acidic glycopeptide fraction (Fraction I) was isolated according to the procedure of Marshall and Neuberger [24]. The glycopeptide fraction was hydrolyzed in 2 M HC1 at 100 °C for 15 min. After cooling, the solution was passed through Amberlite CG-4B (2 × 15 cm) column in the O H - form. The column was eluted with deionized water (150 ml) and the p H of the aqueous effluent was adjusted to 5.6 with 3 M HCI. The solution was lyophilized. The residue was taken up in 1 ml of water and placed in Dowex 50-X4 (200-400 mesh) in the H ÷ form (1 x 4 cm) together with 2 x 1 ml water washings. After washing the column with 80 ml of water, elution was carried out with aqueous HC1 gradient (100 ml water and 100 ml 2 M HC1). Each 3 ml fraction was analyzed for ninhydrin-positive [24] material (Fig. 6). Paper chromatography for isolating N-acetylglucosamine-aspartic acid complex was carried out on Whatman No. 1 paper by the descending technique and the solvent system used was butanol/acetic acid/water (4:1:5) as described by Spiro [18]. The paper was stained with ninhydrin reagent as described by Marshall and Neuberger [24]. The individual components on the paper were located with the help of
96 ninhydrin-stained guide strips and eluted with 0.1 M acetic acid. After lyophilization, the residues were hydrolyzed for 4 h in an evacuated sealed tube in 4 M HC1 at 105 °C for the determination of amino acids and hexosamines [24].
High voltage paper electrophoresis High voltage paper electrophoresis (CAMAC HVE System, U.S.A.) of the pronase-digested peptides (Fraction II) of the 36 000 molecular weight glycoprotein was carried out at 3500 V (70 mA) for 45 min. The solvent system used was acetic acid/formic acid/water (10:5:8). The peptides were dissolved in the same solvent system and spotted on the paper. The peptides which moved toward the cathode were located by ninhydrin reagent and with the help of ninhydrin-stained guide strip, the peptides were eluted from paper with 0.1 M acetic acid. The solution was lyophilized. The residues were hydrolyzed in 6 M HC1 in an evacuated sealed tube under nitrogen at 110 °C for 24 h for the analysis of amino acid.
Amino-terminal sequence analysis of the 18 000 molecular weight CNBr peptide Partial amino-terminal sequence analysis on the 18 000 molecular weight CNBr peptide was done manually according to the sodium dodecyl sulfate-dansylEdman degradation method as described by Weiner et al. [14]. RESULTS The intact glycoprotein was separated from the lavage material by Bio-Gel P-200 column chromatography, as seen in Fig. 1. The third peak contained the purified glycoprotein. The second peak contained the 69 000 and 62 000 molecular weight proteins, as described before [4, 5]. The purified glycoprotein showed a single band on sodium dodecyl sulfate-acrylamide gel electrophoresis (Fig. 4A) with a mobility corresponding to molecular weight 36 000. The molecular weight estimation of the native glycoprotein on sodium dodecyl
36 000
0.5
=E 0.4 69000 + 62000
~Z 0.3 .,
