Biochimica et Biophysica Acta, 439 (1976) 82-94

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 37386 A C O M P L E X - F O R M I N G G L Y C O P R O T E I N H E T E R O G E N E O U S IN C H A R G E AND P R E S E N T IN H U M A N PLASMA, URINE, A N D CEREBROSPINAL FLUID

LARS TEJLER and ANDERS O. GRUBB Department of Clinical Chemistry, University of Lund, Malm~ General Hospital, S-214 Ol Malm6 (Sweden}

(Received October 30th, 1975)

SUMMARY A glycoprotein from the urine of one healthy individual was purified by ultrafiltration, ion-exchange chromatography, gel chromatography and immunosorption. The protein contained only one polypeptide chain with an approximate molecular weight of 31 000 and was associated with a brown colour which did not disappear even after total reduction and alkylation of the protein followed by dialysis in 6 M guanidine hydrochloride. The protein appeared homogeneous on sodium dodecyl sulphate-polyacrylamide electrophoresis and gel chromatography. It had only one N-terminal amino acid sequence, Gly-Pro, and gave only one precipitate with a polyvalent antiserum but was found to be very heterogeneous on agarose gel electrophoresis and on isoelectric focusing. Desialylation of the protein failed to alter this heterogeneity. An electroimmunoassay system was designed to measure the amount of the protein in normal human plasma, urine, and cerebrospinal fluid where the mean concentrations were found to be about 100, 10 and 0.3 mg/1, respectively. The protein was found to occur in normal plasma and urine as free monomers and dimers and as complexes with IgA and albumin.

INTRODUCTION This report describes the purification from normal urine of a glycoprotein which normally occurs in human plasma as complexes with other proteins as well as in the free form. Some physicochemical properties of this electrophoretically heterogeneous protein are also reported. This complex-forming protein heterogeneous in charge is tentatively called protein HC*. MATERIALS AND METHODS Celite Hyflo Super-Cel (Johns-Manville) was obtained through KEBO AB, Abbreviation: SDS, sodium dodecyl sulphate. Protein HC: Human complex-forming glycoprotein, heterogeneous in charge.

83 Stockholm, Sweden. DEAE-Sephadex A-50 and Dextran T 10" (Pharmacia Fine Chemicals AB, Uppsala, Sweden) Ultrogel AcA 54 and ampholytes (LKB-produkter AB, Bromma, Sweden), and agarose (Miles-Seravac, Maidenhead, England) were used. Sodium dodecyl sulphate, acrylamide and N,N'-methylene bisacrylamide, dithiothreitol, dansyl chloride, trifluoroacetic acid, reference amino acids, and polyamide layer sheets were obtained from British Drug Houses Ltd, Poole, England, and phenylisothiocyanate, butylacetate and pyridine (Sequanal grade) from Pierce, Rockford, Ill. Guanidine hydrochloride ("Ultra-pure") and Coomassie Brilliant Blue R 250 was purchased from Schwarz/Mann, Orangeburg, N.Y. Complete Freund's Adjuvant was from Difco Laboratories, Detroit, Mich. All other chemicals were of reagent grade. Human serum albumin was purchased from KABI AB, Stockholm, Sweden. Rabbit antihuman normal urine protein immunoglobulins were obtained from Dakopatts A/S, Copenhagen, Denmark. The other antisera used had earlier been produced at the laboratory. Urine collection. For the purification of protein HC, urine of one healthy individual was collected in plastic bottles that contained sodium azide as a preservative (final concentration 0.2 g/l). The bottles were kept at 4 °C during each 24 h period of collection and then frozen and stored at --25 °C until used. Samples of urine, plasma and serum. These were obtained from healthy volunteers and from patients with chronic renal failure maintained on haemodialysis. Cerebrospinal fluid. This was obtained from patients undergoing diagnostic encephalography. The protein content of each specimen was analysed by the procedure of Lowry et al. [1] with human serum albumin as the standard protein and those with less than 750 mg/1 were pooled. The pooled cerebrospinal fluid was kept at --25 °C until used. Dialysis. Union Carbide 20/32 inch tubing was used. Concentration. Cerebrospinal fluid was concentrated by pressure ultrafiltration using an Amicon Diaflo cell with an UM 2 membrane (nominal retention limit about 1000 daltons). Urine was concentrated, after filtering through Celite Hyflo Super-Cel, by ultrafiltration on a Hollow Fiber Dialyser Concentrator DC 2 (Amicon) equipped with a H1DP 10 capillary filter (nominal retention limit around 10 000 daltons). The procedure used was that recommended by the manufacturer. Both concentration procedures were carried out below 6 °C. Ion-exchange and gel chromatography. The manufacturer's instructions were followed. Immunosorption. An antialbumin immunosorbent was prepared from rabbit antiserum by the glutaraldehyde procedure of Avrameas and Ternynck [2] and packed in a column. Antiserum. This was raised in rabbits by the following immunization procedure: 3 mg of the purified protein HC was dissolved in 1.5 ml of 0.15 M NaC1 and emulsified with 1.5 ml complete Freund's adjuvant. The emulsion was injected into several subcutaneous sites, and was divided in equal parts between 3 rabbits. Identical

* Dextran T 10: The Pharmacia dextran fraction with a mean molecular weight of 10000 prepared from hydrolyzeddextran by repeated partial precipitation.

84 amounts of antigen were used as booster injections 2 weeks later, after which the rabbits were bled every second week. Analytical agarose gel electrophoresis. The procedure of Johansson was used [3]. Crossed immunoelectrophoresis. The system described by Ganrot [4] was used. In some cases the intermediate gel technique of Axelsen [5] was adopted. Immunoelectrophoresis. This was performed in the microsystem of Scheidegger [6]. Electroimmunoassay of protein HC. This was carried out as described by Laurell [7]. Various concentrations of isolated protein HC in 0.15 M NaC1 were used as standards. Antiserum was used at a concentration of about 0.5 ~ . 10 V/cm were applied for 8 h. Electroimmunoassay of albumin. This was carried out as described by Laurell [7]. SDS-polyacrylamide gel electrophoresis. The system of Weber and Osborn was used [8]. The proteins were heated to 90 °C for 1 min, before they were applied to the gel and sometimes mercaptoethanol was added to the sample buffer to a final concentration of 1 ~ (w/v). Isoelectric focusing. Isoelectric focusing was performed in polyacrylamide gel slabs as described by Jeppsson and Berglund [9] wilhout urea in the gel. The completion of the focusing procedure was controlled by awaiting the fusion of two samples of a hemoglobin marker, which had been applied separately near the anode, and the cathode. The pH-gradient of the gel was analysed as described by Salaman and Williamson [10]. Amino acid analysis. Lyophilised, salt free samples were hydrolysed in 6 M HC1 in evacuated, sealed Pyrex tubes at 110 °C for 24 and 72 h. The analyses of the hydrolysates were performed with the two-column system of Spackman et al. [11] on a JEOL Model JLC-5 AH automatic amino acid analyser with the JEOL resin RC 1. Half-cystine was determined as cysteic acid after performic acid oxidation [12]. Amino acid sequence determination. Dansyl-monitored Edman degradations were performed in buffers containing SDS [13] and the dansyl amino acids identified by chromatography on polyamide sheets [14], as outlined by Hartley [15]. Carbohydrate analyses. The amount of neutral hexoses was determined by the anthrone reaction [16] with galactose as standard. Sialic acid was determined after hydrolysis with 0.05 M sulphuric acid at 80 °C for 1 h with the thiobarbituric acid assay [17]. Desialylation of protein HC. Neuraminidase (EC 3.2.1.18) isolated from culture filtrate of Vibrio cholerae was purchased from Behringwerke AG, Marburg-Lahn, G.F.R. 6 mg protein HC was dissolved in 150 /~1 0.05 M sodium acetate buffer, pH 5.5, with 9 mg NaC1 and 1 mg CaC12 per ml and containing 75 units of neuraminidase. The solution was incubated at 37 °C and small aliquots were removed serially, mixed with solutions that contained EDTA in excess and immediately frozen at --70 °C until just before analysis. Total reduction and alkylation. 1.0 mg of protein HC was dissolved in 2.0 ml 2 M Tris. HC1, pH 8.6, that contained 6 M guanidine hydrochloride. After addition of dithiothreitol to a final concentration of 35 mM, the mixture was incubated at 50 °C in a stoppered tube for 2 h, cooled to room temperature, before iodoacetic acid was

85 added to a final concentration of 80 mM. Excess reagents were removed by dialysis against the above-mentioned buffer. Absorption and fluorescence spectra. The absorption spectrum of a solution of protein HC (0.5 mg/ml) in 2 M Tris-HCI, pH 8.6, containing 6 M guanidine hydrochloride was recorded at ambient temperature with the use of a double-beam instrument (Perkin-Elmer 402, Ultraviolet-Visible Spectrophotometer) and with the guanidine-containing buffer as the blank solution. Fluorescence excitation and emission spectra of solutions of protein HC (0.5 mg/ml) in distilled water were recorded with the use of a Perkin-Elmer MPF-3 Fluorescence Spectrophotometer. RESULTS

Purification 37 litres of urine concentrated to a final volume of 300 ml was dialysed against 0.1 M Tris. HC1, pH 8.0, with 2 mM CaC12, cleared by centrifugation and subjected to ion-exchange on a DEAE-Sephadex A-50 column that had been previously equilibrated with the same buffer (Fig. 1). Fractions which contained protein HC were identified by antiserum and pooled as shown in Fig. l, dialysed against distilled water and lyophilised. The lyophilised proteins were further separated by three chromatographic steps on Ultrogel AcA 54 in 0.05 M NH4HCO3. Fractions that contained protein HC were pooled and lyophilised between each chromatographic run (Fig. 1). The protein lyophilised from the third chromatographic gel run was dissolved in 10 ml of distilled water, cleared by centrifugation, and 1 ml 0.1 M phosphate buffer, pH 7.4, was added before passage through a column which contained antialbumin immunosorbent. The effluent in which no albumin could be detected by the electroimmunoassay was dialysed against distilled water and lyophilised. The yield was 55 mg which corresponds to a recovery of 10~ as estimated from the electroimmunoassay of the original urine sample.

Criteria of purity The purified protein gave only one precipitation arc on immunoelectrophoresis with the use of a polyvalent antiserum against normal human urinary proteins and only one fraction when analysed by SDS-polyacrylamide electrophoresis. The glycoprotein had only one N-terminal residue, glycine, and only one penultimate residue, proline, as revealed by chromatography of the dansyl amino acids on polyamide sheets.

Carbohydrate analysis Protein HC contained 11.0 ~o neutral hexoses (mean of 4 determinations) as determined by the anthrone reaction and 11.4 ~ sialic acid (mean of 4 determinations) as estimated by the thiobarbituric acid assay.

Number of polypeptide chains and molecular weight SDS-polyacrylamide electrophoresis of the unreduced and reduced protein gave in both instances only one narrow band. By use of serum albumin, H- and L-

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A complex-forming glycoprotein heterogeneous in charge and present in human plasma, urine, and cerebrospinal fluid.

Biochimica et Biophysica Acta, 439 (1976) 82-94 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 37386 A C O M P...
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