152
Biochimica et Biophysica Acta, 1034 (1990) 152-156 Elsevier
BBAGEN 23298
Human seminal plasma Zn-a2-glycoprotein *" Its purification and properties as compared with human plasma Zn-az-glycoprotein Iwao Ohkubo 1, Masaaki Niwa 1, Akira Takashima 2, N a o k o Nishikimi 3, Shinsei Gasa 4 and Makoto Sasaki 1 1 Department of Biochemistry, 2 Department of Dermatology and 3 Department of Obstetrics and Gynecology, Nagoya City University Medical School, Nagoya and 4 Biochemistry Laboratory, Cancer Institute, Hokkaido University School of Medicine, Sapporo (Japan) (Received 1 October 1989)
Key words: Zinc-a2-glycoprotein; Glycoprotein; Seminal plasma; Amino acid sequence; Carbohydrate content; (Human)
On the basis of the datum that the level of Zn-a2-glycoprotein (Zna2gp) in human seminal plasma was about 6-times higher than that in adult serum, Znazg p was purified from fresh human seminal plasma approx. 70-fold with 60% yield over seminal plasma by DEAE-Sephacel, Zn-chelate Sepharose 4B and DEAE-5PW column chromatographies. The molecular weight of seminal plasma Znazgp was 50000 on Superose column chromatography, and 40 500 and 41500 on SDS-polyaerylamide gel electrophoresis in the absence and presence of fl-mercaptoethanol, respectively. Plasma Zna2g p is a glycoprotein, while the protein from seminal plasma does not contain carbohydrate. The amino acid sequence of the first 17 residues of seminal plasma Zna2gp was Glu-Asn-Gin-Asp-Giy-Asn-Tyr-Ser-Leu-Thr-Tyr-IleTyr-Thr-Gly-Leu-Ser. This sequence was completely identical with the amino acid residues from Glu-2 to Ser-18 in the N-terminal amino acid sequence of plasma Zna2g p. These data suggest that both Zna2gps in plasma and seminal plasma may be expressed from one gene, but their posttranslational modifications are different.
Introduction Zn-a2-glycoprotein (Zna2gp) is a glycoprotein with a low molecular weight (3 S) of 38 000-41000 in normal plasma (or serum) [1]. On the basis of electrophoresis in the a2-region and precipitation by zinc ions, it was designated as Znazg p [1]. The physicochemical properties of Znotzgp have been clarified [1,2]. Shibata et al. [3] indicated the possibility that this protein plays an important role as a carrier protein of nephritogenic renal glycoprotein(s). Recently, Araki et al. [4] showed that the amino acid sequence of Znazg p is similar to those of major histocompatibility antigens (HLA class I-a chain and HLA class II), and also
* In this paper, we have tentatively used Zn-a2-glycoprotein (Znazgp) for the purified human seminal plasma protein which proved not to be a glycoprotein. Abbreviations: fl-ME, fl-mercaptoethanol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; HPLC, high-performance liquid chromatography; Zna2g p, Zn-a2-glycoprotein. I. Ohkubo, Department of Biochemistry, Nagoya City University Medical School, Mizuho-ku, Nagoya, Aichi 467, Japan.
Correspondence:
speculated that the protein has a role in the expression of the immune response. However, the physiological role and pathological significance are still unknown. During the quantitative determination of Zna2g p in normal human seminal plasma, we found that the level of Zna2g p in seminal plasma was 6-times higher than that in adult serum. In this paper, we describe a novel procedure for the isolation of Zna2gp from human seminal plasma and the characterizations of this protein in comparison with human plasma Zna2g p. Materials Human seminal fluid used for the purification of Zna2g p was obtained from healthy Japanese volunteers, and seminal plasma was separated from spermatozoa by centrifugation at 10000 rpm for 15 min at room temperature. DEAE-Sephacel, Sephacryl S-300 HR, Sepharose 4B and standard proteins for molecular weight determination were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Agarose L and anti-human Z n a 2 g p antiserum were products of Behringwerke (Marburg, FRG). Zinc-chelate Sepharose 4B was prepared by the method of Porath et al. [5]. All other chemicals were of analytical grade.
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
153 Methods
Purification of Zn-ct2-glycoprotein from human seminal plasma All steps of the purification procedure were carried out at 4°C, unless otherwise specified. Concentration of Zna2gp was determined by single radial immunodiffusion in comparison with a simultaneous standard of purified plasma Zna2g p which was calculated by extinction coefficient (E280n 1% m _- 18) [1]. Step (1) Dialysis. Seminal plasma was dialyzed overnight against 20 mM sodium phosphate buffer (pH 7.2). The seminal plasma was centrifuged at 10 000 rpm for 30 min. The supernatant obtained was used for subsequent step. Step (2). DEAE-Sephacel column chromatography. The supernatant was applied at a flow rate of 25 m l / h to a DEAE-Sephacel column (1.6 x 24 cm) equilibrated with 20 mM sodium phosphate buffer (pH 7.2). After application of the sample, the column was washed thoroughly with the equilibration buffer and bound proteins were eluted by a linear gradient formed by 250 ml of 20 mM sodium phosphate buffer (pH 7.2) and 250 ml of the same buffer containing 0.5 M NaC1. Fractions containing Znazgp were collected and dialyzed overnight against 20 mM Tris-HC1 buffer (pH 7.5)/0.2 M NaC1.
Step (3). Zn-chelate Sepharose 4B column chromatography. The dialyzate was applied at a flow rate of 30 m l / h to a Zn-chelate Sepharose 4B column (2 x 21 cm) equilibrated with 20 mM Tris-HC1 (pH 7.5)/0.2 M NaC1. After application of the sample, the column was washed thoroughly with the equilibration buffer, and bound proteins were eluted with 0.2 M EDTA in the above buffer. The pass-through fraction (second peak) containing Znctzgp was collected and dialyzed overnight against 20 mM Tris-HC1 buffer (pH 7.5).
Step (4). TSK gel DEAE-5PW column chromatography. One-fifth of the dialyzate was applied at a flow rate of 0.8 ml/min to a TSK gel DEAE-5PW column using FPLC system, equilibrated with 20 mM Tris-HC1 buffer (pH 7.5). After application of the sample, the column was washed with the same buffer. A stepwise gradient was applied to the column as follows: at first a linear gradient of 0.8 ml of the same buffer and 0.8 ml of the same buffer containing 0.1 M NaC1, followed by a second linear gradient of 8 ml of the same buffer containing 0.1 M NaC1 and 8 ml of the same buffer containing 0.3 M NaC1. Zna2g p eluted as the major peak in the second gradient. The remaining portion of the dialyzate was chromatographed in the same manner. The preparation was used for subsequent studies.
Purification of Zn-a2-glycoprotein from human plasma Human plasma Znazg p was purified from fresh citrated human plasma as described previously [2].
Amino acid analysis Amino acid composition of seminal plasma Zna2g p was determined as described previously [2]. Tryptophan was determined spectrophotometrically [6].
Carbohydrate analysis The carbohydrate composition of human plasma Zna2gp was determined as described previously [2,7,8]. For precise determination of the carbohydrate composition of human seminal plasma Zna2gp, two different methods (an alditol acetate method, in which neutral sugar and hexosamine are converted to their alditol acetates, and a fluorescence-labeled method, in which the reducing end of sugar chains are aminated with 2-aminopyridine) described previously by Hakomori [9] and Hase et al. [10,11] were employed.
Amino acid sequence Automated Edman degradations on both Znot2gps (50 /~g) were performed on an Applied Biosystems Model 477A Protein Sequencer. Phenylthiohydantoin amino acids were identified by high-performance liquid chromatography (Applied Biosystems 120A analyzer).
Polyacrylamide gel electrophoresis SDS-polyacrylamide slab gel electrophoresis was carried out by the method of Laemmli [12].
Immunodiffusion Double immunodiffusion and single radial immunodiffusion were performed by the methods of Ouchterlony [13] and Mancini [14], respectively.
Preparation of antiserum for human plasma Zn-ote-glycoprotein Antiserum for human plasma Znot2gp was prepared as described previously [2]. Results and Discussion
Znet2gp was purified from human seminal plasma by chromatography on DEAE-Sephacel, Zn-chelate Sepharose 4B and DEAE-5PW columns in FPLC system (Fig. 1A-C). A typical purification procedure of Zna2g p is summarized in Table I. Human seminal plasma Zn~2gp was finally purified approx. 68-fold with 60% yield over seminal plasma, and the protein gave a single band on SDS-PAGE in the absence and presence of /3-mercaptoethanol (fl-ME) (Fig. 2). The overall yield of Znot2g p w a s approx. 26.5 mg from 100 ml of human seminal plasma. On the other hand, plasma Z n ~ 2 g p w a s purified about 670-fold with 18% yield by the procedure previously reported [2], and the overall yield of plasma Znct2gp was only 2.5 mg from 500 ml of fresh human plasma.
154 The molecular weight of the protein from human seminal plasma was estimated to be 50 000 on Superose column chromatography in FPLC system. On the other hand, the molecular weight of Zna2gp purified from
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Biochimica et Biophysica Acta, 1034 (1990) 152-156 Elsevier
BBAGEN 23298
Human seminal plasma Zn-a2-glycoprotein *" Its purification and properties as compared with human plasma Zn-az-glycoprotein Iwao Ohkubo 1, Masaaki Niwa 1, Akira Takashima 2, N a o k o Nishikimi 3, Shinsei Gasa 4 and Makoto Sasaki 1 1 Department of Biochemistry, 2 Department of Dermatology and 3 Department of Obstetrics and Gynecology, Nagoya City University Medical School, Nagoya and 4 Biochemistry Laboratory, Cancer Institute, Hokkaido University School of Medicine, Sapporo (Japan) (Received 1 October 1989)
Key words: Zinc-a2-glycoprotein; Glycoprotein; Seminal plasma; Amino acid sequence; Carbohydrate content; (Human)
On the basis of the datum that the level of Zn-a2-glycoprotein (Zna2gp) in human seminal plasma was about 6-times higher than that in adult serum, Znazg p was purified from fresh human seminal plasma approx. 70-fold with 60% yield over seminal plasma by DEAE-Sephacel, Zn-chelate Sepharose 4B and DEAE-5PW column chromatographies. The molecular weight of seminal plasma Znazgp was 50000 on Superose column chromatography, and 40 500 and 41500 on SDS-polyaerylamide gel electrophoresis in the absence and presence of fl-mercaptoethanol, respectively. Plasma Zna2g p is a glycoprotein, while the protein from seminal plasma does not contain carbohydrate. The amino acid sequence of the first 17 residues of seminal plasma Zna2gp was Glu-Asn-Gin-Asp-Giy-Asn-Tyr-Ser-Leu-Thr-Tyr-IleTyr-Thr-Gly-Leu-Ser. This sequence was completely identical with the amino acid residues from Glu-2 to Ser-18 in the N-terminal amino acid sequence of plasma Zna2g p. These data suggest that both Zna2gps in plasma and seminal plasma may be expressed from one gene, but their posttranslational modifications are different.
Introduction Zn-a2-glycoprotein (Zna2gp) is a glycoprotein with a low molecular weight (3 S) of 38 000-41000 in normal plasma (or serum) [1]. On the basis of electrophoresis in the a2-region and precipitation by zinc ions, it was designated as Znazg p [1]. The physicochemical properties of Znotzgp have been clarified [1,2]. Shibata et al. [3] indicated the possibility that this protein plays an important role as a carrier protein of nephritogenic renal glycoprotein(s). Recently, Araki et al. [4] showed that the amino acid sequence of Znazg p is similar to those of major histocompatibility antigens (HLA class I-a chain and HLA class II), and also
* In this paper, we have tentatively used Zn-a2-glycoprotein (Znazgp) for the purified human seminal plasma protein which proved not to be a glycoprotein. Abbreviations: fl-ME, fl-mercaptoethanol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; HPLC, high-performance liquid chromatography; Zna2g p, Zn-a2-glycoprotein. I. Ohkubo, Department of Biochemistry, Nagoya City University Medical School, Mizuho-ku, Nagoya, Aichi 467, Japan.
Correspondence:
speculated that the protein has a role in the expression of the immune response. However, the physiological role and pathological significance are still unknown. During the quantitative determination of Zna2g p in normal human seminal plasma, we found that the level of Zna2g p in seminal plasma was 6-times higher than that in adult serum. In this paper, we describe a novel procedure for the isolation of Zna2gp from human seminal plasma and the characterizations of this protein in comparison with human plasma Zna2g p. Materials Human seminal fluid used for the purification of Zna2g p was obtained from healthy Japanese volunteers, and seminal plasma was separated from spermatozoa by centrifugation at 10000 rpm for 15 min at room temperature. DEAE-Sephacel, Sephacryl S-300 HR, Sepharose 4B and standard proteins for molecular weight determination were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Agarose L and anti-human Z n a 2 g p antiserum were products of Behringwerke (Marburg, FRG). Zinc-chelate Sepharose 4B was prepared by the method of Porath et al. [5]. All other chemicals were of analytical grade.
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
153 Methods
Purification of Zn-ct2-glycoprotein from human seminal plasma All steps of the purification procedure were carried out at 4°C, unless otherwise specified. Concentration of Zna2gp was determined by single radial immunodiffusion in comparison with a simultaneous standard of purified plasma Zna2g p which was calculated by extinction coefficient (E280n 1% m _- 18) [1]. Step (1) Dialysis. Seminal plasma was dialyzed overnight against 20 mM sodium phosphate buffer (pH 7.2). The seminal plasma was centrifuged at 10 000 rpm for 30 min. The supernatant obtained was used for subsequent step. Step (2). DEAE-Sephacel column chromatography. The supernatant was applied at a flow rate of 25 m l / h to a DEAE-Sephacel column (1.6 x 24 cm) equilibrated with 20 mM sodium phosphate buffer (pH 7.2). After application of the sample, the column was washed thoroughly with the equilibration buffer and bound proteins were eluted by a linear gradient formed by 250 ml of 20 mM sodium phosphate buffer (pH 7.2) and 250 ml of the same buffer containing 0.5 M NaC1. Fractions containing Znazgp were collected and dialyzed overnight against 20 mM Tris-HC1 buffer (pH 7.5)/0.2 M NaC1.
Step (3). Zn-chelate Sepharose 4B column chromatography. The dialyzate was applied at a flow rate of 30 m l / h to a Zn-chelate Sepharose 4B column (2 x 21 cm) equilibrated with 20 mM Tris-HC1 (pH 7.5)/0.2 M NaC1. After application of the sample, the column was washed thoroughly with the equilibration buffer, and bound proteins were eluted with 0.2 M EDTA in the above buffer. The pass-through fraction (second peak) containing Znctzgp was collected and dialyzed overnight against 20 mM Tris-HC1 buffer (pH 7.5).
Step (4). TSK gel DEAE-5PW column chromatography. One-fifth of the dialyzate was applied at a flow rate of 0.8 ml/min to a TSK gel DEAE-5PW column using FPLC system, equilibrated with 20 mM Tris-HC1 buffer (pH 7.5). After application of the sample, the column was washed with the same buffer. A stepwise gradient was applied to the column as follows: at first a linear gradient of 0.8 ml of the same buffer and 0.8 ml of the same buffer containing 0.1 M NaC1, followed by a second linear gradient of 8 ml of the same buffer containing 0.1 M NaC1 and 8 ml of the same buffer containing 0.3 M NaC1. Zna2g p eluted as the major peak in the second gradient. The remaining portion of the dialyzate was chromatographed in the same manner. The preparation was used for subsequent studies.
Purification of Zn-a2-glycoprotein from human plasma Human plasma Znazg p was purified from fresh citrated human plasma as described previously [2].
Amino acid analysis Amino acid composition of seminal plasma Zna2g p was determined as described previously [2]. Tryptophan was determined spectrophotometrically [6].
Carbohydrate analysis The carbohydrate composition of human plasma Zna2gp was determined as described previously [2,7,8]. For precise determination of the carbohydrate composition of human seminal plasma Zna2gp, two different methods (an alditol acetate method, in which neutral sugar and hexosamine are converted to their alditol acetates, and a fluorescence-labeled method, in which the reducing end of sugar chains are aminated with 2-aminopyridine) described previously by Hakomori [9] and Hase et al. [10,11] were employed.
Amino acid sequence Automated Edman degradations on both Znot2gps (50 /~g) were performed on an Applied Biosystems Model 477A Protein Sequencer. Phenylthiohydantoin amino acids were identified by high-performance liquid chromatography (Applied Biosystems 120A analyzer).
Polyacrylamide gel electrophoresis SDS-polyacrylamide slab gel electrophoresis was carried out by the method of Laemmli [12].
Immunodiffusion Double immunodiffusion and single radial immunodiffusion were performed by the methods of Ouchterlony [13] and Mancini [14], respectively.
Preparation of antiserum for human plasma Zn-ote-glycoprotein Antiserum for human plasma Znot2gp was prepared as described previously [2]. Results and Discussion
Znet2gp was purified from human seminal plasma by chromatography on DEAE-Sephacel, Zn-chelate Sepharose 4B and DEAE-5PW columns in FPLC system (Fig. 1A-C). A typical purification procedure of Zna2g p is summarized in Table I. Human seminal plasma Zn~2gp was finally purified approx. 68-fold with 60% yield over seminal plasma, and the protein gave a single band on SDS-PAGE in the absence and presence of /3-mercaptoethanol (fl-ME) (Fig. 2). The overall yield of Znot2g p w a s approx. 26.5 mg from 100 ml of human seminal plasma. On the other hand, plasma Z n ~ 2 g p w a s purified about 670-fold with 18% yield by the procedure previously reported [2], and the overall yield of plasma Znct2gp was only 2.5 mg from 500 ml of fresh human plasma.
154 The molecular weight of the protein from human seminal plasma was estimated to be 50 000 on Superose column chromatography in FPLC system. On the other hand, the molecular weight of Zna2gp purified from
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