Purification and characterization of the P-80 glycoprotein from human ...

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Biochem. J. (1988) 256, 351-356 (Printed in Great Britain)

351

Purification and characterization of the P-80 glycoprotein from human brain Nigel GIRGRAH,* Tony F. CRUZ,* Michelle LETARTEt and Mario A. MOSCARELLO*t Departments of *Biochemistry and tlmmunology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8

A glycoprotein antigen was purified from human brain by immunoaffinity chromatography using the 44D 10-monoclonal IgG, and its chemical nature was investigated. The yield of antigen was estimated at 91 Qo and a 4340-fold purification was obtained relative to the white-matter homogenate. The antigen preparation from brain was further purified by preparative SDS/polyacrylamide-gel electrophoresis (PAGE) to obtain a glycoprotein with an Mr of 80000 consisting of a single polypeptide. Amino acid analyses revealed a composition which was high in acidic and neutral amino acids, and low in basic residues. The presence of both glucosamine and galactosamine suggested that the glycoprotein contained both N- and 0-linked glycans. Neutral sugar analyses showed that fucose, galactose and mannose were present. An assay for sialic acid determined that there were approximately 20 mol of sialic acid per mol of glycoprotein. Chemical cleavage of oligosaccharides by trifluoromethanesulphonic acid followed by SDS/PAGE showed that carbohydrate accounted for 25000 of the 80000-Mr glycoprotein.

INTRODUCTION In a previous investigation it was rep'orted that three monoclonal antibodies (mAbs), 44H4, 44D7 and 44D10, generated against a non-B acute lymphoblastic leukaemic cell line, HOON, cross-reacted with homogenates from human brain tissue (Quackenbush et al., 1985). While antigens reactive with mAbs 44D7 and 44H4 were found to be present in both grey and white matter of human brain, the antigen reactive with mAb 44D10 was detected only in the white matter. Detergent solubilization of white matter followed by immunoaffinity chromatography of the extract, and subsequent radiolabelling and immunoprecipitation of the eluted protein, revealed that mAb 44D10 was directed against a determinant on a glycoprotein of Mr 80000, as -determined by SDS/polyacrylamide-gel electrophoresis (PAGE) (Quackenbush et al., 1985; Cruz et al., 1985). Competitive inhibition studies (Letarte et al., 1985) showed that the antigen reactive with mAb 44D10 was homologous to antigens reactive with previously reported monoclonal antibodies, namely the F10.44.2 antigen first described in human T lymphocytes, granulocytes and in brain (Dalchau et al., 1980; McKenzie et al., 1982), the A3D8 antigen present on most human erythrocytes (Telen et al., 1983, 1984, 1985), and A1G3 antigen reported on mature human thymocytes (Haynes et al., 1983). These antibodies were shown to define three distinct epitopes on the molecule (Letarte, 1986). Antigens reactive with mAb F10,44.2 and related antibodies are now classified in the CDw44 cluster (Cobbold et al., 1987). Recent observations have established that the phagocyte glycoprotein-1 (pgp-1) antigen, abundant on macrophages, monocytes and polymorphonuclear cells, also belongs to the CDw44 cluster and is highly related to, if not identical, with the P-80 glycoprotein (Omary et al., 1988; Mackay et al., 1988).

In an earlier report, we showed that the 44D10 antigen was increased in concentration in white matter and was present in low levels in grey matter of brains ofindividuals over the age of 55 years (Cruz et al., 1985). Concentrations of the antigen in brains from individuals who died of neurological disease were also determined (Cruz et al., 1986). The 44D10 antigen was not significantly elevated in brains of individuals who had died with Alzheimer's, Parkinson's or Huntington's diseases when compared with age-matched normal control brains (sudden-death casualties); however, the antigen was detected in grey matter and at elevated levels in white matter of brains of victims of multiple sclerosis. In the present study, we report the purification and partial characterization from human brain white matter of the glycoprotein reactive with monoclonal antibody 44D10 and designated P-80.

MATERIALS AND METHODS Preparation of immunoadsorbent column mAb 44D10 was obtained by the immunization of Balb/c mice with cells from the non-T, non-B lymphoblastic leukaemic cell line, HOON (Okamura et al., 1984), and fusion of the Balb/c spleen cells with the SP2/0-Agl4 non-secretory myeloma cells (Quackenbush & Letarte, 1985). The IgG fraction was obtained by 45 % ammonium sulphate precipitation of the hybridoma culture supernatant followed by a further fractionation by anion-exchange chromatography (DEAE-Sephadex A-80) of the precipitate. The purified IgG fraction was then coupled to CNBr-activated Sepharose 4B. Fractionation of human white matter Fractionation of white matter was an extension of a previously described procedure (Lowden et al., 1966).

Abbreviations used: mAb, monoclonal antibody; PAGE, polyacrylamide-gel electrophoresis; TFMS, trifluoromethanesulphonic acid; RAM, rabbit anti-mouse; PMSF, phenylmethanesulphonyl fluoride; RAM-IgG, rabbit IgG anti-mouse IgG; RAM-Fc, F(ab')2 pepsin fragment of rabbit IgG produced against the purified papain Fc fragment of mouse IgG and purified by affinity to mouse IgG. t To whom correspondence and reprint requests should be addressed.

Vol. 256

352

N. Girgrah and others Homogenate (0.25 M-sucrose)

Purification of the P-80 glycoprotein by immunoaffinity

chromatography Centrifuge 100,000 g, 90 min

The soluble extract was first passed sequentially a non-immune mouse IgG-Sepharose 4B column and then through the mAb 44D10-Sepharose 4B column. The columns were washed in 50 mM-Tris, pH 8.0, containing 0.6 % cholate, 0.04 % NaN3, 0.001 MPMSF, and specifically-bound protein was eluted from the mAb 44D10-Sepharose 4B column with 50 mmdiethylamine, pH 11.2, containing 0.6 % cholate and neutralized immediately with 0.5 M-Tris, pH 7.5. The antigen was concentrated 5- to 6-fold by Amicon pressure filtration and 10 % (w/v) SDS was added to give a final concentration of 1 % SDS. The preparation was heated for 10 min at 37 °C, dialysed successively against 0.1 % SDS for 16 h, 0.05 % SDS for 3 h and 0.02 % SDS for 3 h. Samples were lyophilized and prepared for SDS/ PAGE (Laemmli, 1970). Immunoabsorbed protein was fractionated by 10 % (w/v) SDS/PAGE, and P-80 glycoprotein was electroeluted from the gel (Iscoeluter apparatus, Isco, Lincoln, NE, U.S.A.). The electroeluate was precipitated in 80 % acetone at -20 °C and residual salts were removed by extraction in acetone/acetic acid/triethylamine/H20 (17:1:1:1, by vol.). Immunoprecipitation of the P-80 glycoprotein Portions of affinity-purified protein (10 ,ug) were labelled with 1211 by using 15 ,ug lodogen (Pierce, Rockford, IL, U.S.A.) (Markwell & Fox, 1978); free iodine was removed by Sephadex G-50 chromatography. Radiolabelled P-80 was immunoprecipitated as follows. Radiolabelled affinity-purified samples were incubated with RAM-IgG-Sepharose (30 min, 4 °C) before incubation with saturating levels of mAb 44D10 (16 h; 4 °C) and RAM-IgG-Sepharose (1 h; 4 °C). Beads were washed three times and eluted with 2 % SDS. Eluates were counted for radioactivity and subjected to 10 % (w/v) SDS/PAGE. Autoradiography was performed using XAR-1 film and DuPont Cronex Hi-Plus intensifying screens (Swanstrom & Shank, 1978). Celular radioimmunoassay The presence of antigen reactive with mAb 44D10 in each of the purification steps was determined by the inhibition of cellular radioimmunoassay as described previously (Letarte et al., 1980; Quackenbush et al., 1985). Briefly, serial dilutions of the brain fractions to be tested (50,1l) in phosphate-buffered saline containing 1 % bovine serum albumin were incubated for 16 h at 4 °C with a 1:300 dilution of mAb 44D10 culture supernatant. After 8 min centrifugation at 10000 g, 25 1dl aliquots, in triplicate, were transferred to microtitre plates and incubated at 4 °C for 2 h with 1 x 106 glutaraldehyde-fixed HOON target cells (25 ,ul). Residual mAb bound to the target cells was detected by incubation at 4 °C for 2 h with 125I-RAM-Fc (25 ng/well; 100000 c.p.m.) (Letarte et al., 1980). The specific activities were calculated in units/mg of protein, 1 unit being defined as the amount of protein required to block 50 % of the antibody from binding to the target cells. The amount of protein in each of the purification steps was determined by the method of Peterson (1977). Amino acid compositions Samples containing 2-5 ,ug of protein were desalted by precipitation in acetone/acetic acid/triethylamine/

through

P2

Si

Centrifuge -100,000 g, 60 min I Myelin

P3 (Solubilize 50 mM-Tris/HCI, 1.5 % Triton X- 1 00, pH 8.0)

Centrifuge 96,000 g, 90 min Detergent-soluble fraction

0 Non - immu mouse IgG

_

I

I 6m." 44D10 mAbSepharose4E

Sepharose

0 0

Elute-bound protein with 50 mM-diethylamine, 0u 0.6% cholate, pH 11.2

Preparativegel electrophoresis

Electroelution

Fig. 1. Isolation of P-80 glycoprotein The flow chart summarizes the isolation and purification of P-80 from a homogenate of human brain white matter.

Briefly, white matter (35 g) dissected from human brain was homogenized in cold 0.25 M-sucrose with a motordriven glass/Teflon homogenizer, diluted to 180 ml and centrifuged as shown in Fig. 1. Fraction P2 was resuspended in 120 ml of 0.88 M-sucrose and divided into 10 ml portions, which were layered with 3 ml of 0.25 Msucrose and centrifuged at 100000 g for 60 min. After centrifugation, the 0.25 M-/0.88 M-sucrose interface was discarded or removed for the preparation of myelin as previously described (Lowden et al., 1966). The pellet, P3, was solubilized in a final concentration of 1.5 % Triton X-100 in 50 mM-Tris/HCl, pH 8.0, containing 0.04 % NaN3 and supplemented with 0.001 M-phenylmethanesulphonyl fluoride (PMSF) for 2 h at 4 'C. The mixture was centrifuged at 100000 g for 60 min to obtain the soluble extract.

1988

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Human brain P-80 glycoprotein

H20 (17:1:1:1, by vol.), dried down under N2 and hydrolysed in HCI vapour for 24 h at 80 'C. Amino acids were derivatized with phenylisothiocyanate and analysed on the Waters Picotag system. Tryptophan was determined by hydrolysis of P-80 in methanesulphonic acid followed by analysis on the Waters Picotag System. Cysteine was determined by reduction and radioalkylation using "C-labelled iodoacetamide as follows. Purified P-80 glycoprotein (10 ,ug) was dissolved in 20 1l of 8 M-urea, pH 8.6 (pH adjusted with 5 % methylamine). k6-Mercaptoethanol was added such that the final concentration was 50 /tM; the solution was flushed with N2 and allowed to react for 4-5 h at room temperature. After the reduction step, 0.2 /tmol of [1"C]iodoacetamide (specific activity 25 ,uCi/,umol) dissolved in 80 1Al of 0.1 M-Tris acetate buffer, pH 8.5, was added. The mixture was again flushed with N2 and allowed to react for 30 min at room temperature. The reduced and alkylated glycoprotein was prepared for amino acid analysis as described earlier. The volume corresponding to carboxymethylcysteine was collected from the amino acid analyser and the radioactivity was determined. From the measured radioactivity the amount of carboxymethylcysteine was calculated, from which the amount of cysteine in the P-80 glycoprotein was computed. Carbohydrate analyses The presence of glucosamine and galactosamine was determined on the Waters Picotag amino acid analyser following hydrolysis of 2-5 ,tg of purified glycoprotein with 4.0 M-HCI at 105 'C for 5 h under vacuum. After derivatization, glucosamine and galactosamine can be readily separated from the amino acids and quantified. From these values the amounts of N-acetylglucosamine and N-acetylgalactosamine, which are both de-Nacetylated during hydrolysis, can be computed. The neutral sugars, fucose, galactose and mannose were determined by the method of Zanetta et al. (1972). Approx. 25 #g of purified glycoprotein was incubated at 80 IC for 20 h in methanolic HCI (1.0 ml anhydrous methanol, 35 #1 acetyl chloride). The sample was dried down under N2 and derivatized with 0.5 ml trifluoroacetic acid at 80 'C for 30 min. The sample was concentrated to Table 1. Purification of a glycoprotein from white matter of human brain by adsorption to mAb 44Dl-lIgGSepharose White matter homogenate was fractionated by sucrosedensity-gradient centrifugation and the 0.88 M pellet, P3, was solubilized and fractionated on mAb 44D10-IgGSepharose. All fractions were assayed for 44D10 activity by inhibition of the cellular radioimmunoassay.

Fraction Homogenate P2 P3

Myelin Triton X-100

Yield Protein Specific activity Purification (%) (mg) (-fold) (units/mg) 100 79.9 86.2 31.2 59.3

extract

Immunoadsorbed 91.0 protein

Vol. 256

2150 1790 1005 816 368

7.7 7.4 14.2 6.3 26.6

0.45 33440

0.96 1.84 3.4

4343

approximately 20 ,ul and injected directly into a Varian Aerograph series g.l.c. analyser. Sugar standards purchased from Sigma were methanolysed in the same way and trifluoroacetic acid derivatives were obtained. These derivatized sugars were used to standardize the g.l.c. Carbohydrate analyses were also done on a Dionex Bio LC, consisting of an anion-exchange column coupled to pulsed amperometric detection. Eluent 1 was 15 mmNaOH and eluent 2 was 200 mM-NaOH. In preparation for the chromatography, the P-80 glycoprotein (30 jig) was hydrolysed in 2 M-trifluoracetic acid at 100 °C for 4-5 h. The trifluoroacetic acid was removed by lyophilization and the residue was suspended in 250 ,1 of h.p.l.c.-grade water of which 100 ,1 was added to the Dionex Carbopac AS-6 column. The monosaccharides were resolved isocratically with eluent 1 in 15 min. The column was then washed with eluent 2 for 5 min. The following pulse potentials were used: E1, 0.05 V; E2 0.60 V; and E39 0.80 V. Sialic acid determination Sialic acid was quantified by the method of Aminoff (1961). Briefly, sialic acid was released from approximately 10 ,tg of glycoprotein by mild acid hydrolysis in 0.2 ml of 0.1 M-H2SO4 at 80 °C for 20 h. Subsequently 0.1 ml of 25 mM-NalO4 was added and the mixture was incubated for 30 min at 37 'C. To destroy excess periodate, 0.1 ml of sodium arsenite (2 % in 0.5 MHCI) was added, and the samples were vortexed until colourless. Then 0.8 ml of thiobarbituric acid (0.1 M) was added, and the samples were boiled for 5-7 min at 100 'C and placed on ice to cool. Acid/butanol (5 % concentrated HCI in n-butanol; 1 ml) was added and the samples were vortexed and allowed to stand at room temperature for 10 min. Samples were then centrifuged at 3000 g for 10 min to separate the aqueous and organic layers. The organic layer (upper) was removed and the absorbance was read at 549 nm. The absorbance values at 549 nm were compared with values obtained by using known quantities of sialic acid standards. Deglycosylation of P-80 with TFMS Gel-purified P-80 was radioiodinated using the lodogen method (Markwell & Fox, 1978). Labelled glycoprotein (5 x 105 c.p.m.) was dried down under N2 and 50 ,1 of TFMS/anisole (9: 1, v/v) was added and was allowed to react for 2 h at 0 'C. The reaction was stopped by the addition of 500 #1 of diethyl ether/pyridine (9: 1, v/v), cooled on acetone/dry ice and the reaction mixture was centrifuged at 10000 g for 10 min. The supernatant was discarded and the pellet was resuspended in 1 % NH4HCO3 and dialysed three times against 1 % NH4HCO3. The sample was recovered by centrifugation at 10000 g for 10 min and was prepared for SDS/PAGE. Samples with or without chemical treatment with TFMS were counted for radioactivity and subjected to 10 % (w/v) SDS/PAGE (Laemmli, 1970). Autoradiography was performed with XAR-1 film and DuPont Cronex Hi-plus intensifying screens. RESULTS Purification of the P-80 glycoprotein from human white matter

Fraction P3 (Fig. 1) was obtained from human white previously described (Quackenbush et al.,

matter as

354

.-:"oi;| 2

1

.....

......

..

....

N. Girgrah and others 10-3xMr

The results are expressed as the mean of each amino acid residue per 100 residues and represent eight independent amino acid analyses of four separate batches of P-80

glycoprotein.

.

94 467

....' mi.':.:.::

Amino acid

Residues/ 100

Asx Thr Ser Glx Pro

13.1 +4.3 6.7+0.6 11.9+ 1.3 10.6+ 1.1 5.4 + 1.0 13.1+ 1.0 6.4+0.7 4.1 +0.5 Undetectable 1.0+0.3 4.3 + 0.5 5.5+1.0 2.1+ 1.0 2.9+0.6 3.2+0.8 2.0+0.4 3.8 +0.6

Gly Ala Val Cys* Met Ile Leu Tyr Phe Lys His Arg

.443

. 30

..f.....

fU=Y=,0" ': '.

Table 2. Amino acid compositions of purified P-SO

t

Fig. 2. SDS/PAGE of purified P-80 glycoprotein Portions of affinity-purified protein were labelled with 125I and a sample (5000 c.p.m.) was fractionated by SDS/ PAGE (10 %, w/v) (lane I). Radiolabelled affinity-purified samples were incubated with RAM-IgG-Sepharose (30 min, 4 °C) before incubation with saturating levels of mAb 44D10 (16 h; 4 °C) and RAM-IgG-Sepharose (I h; 4 C)Y. Beads were washed three times and eluted with 2 % SDS. A portion of the eluate (5000 c.p.m.) was subjected to SDS/PAGE (10%, w/v) (lane 2). Autoradiography was performed using XAR- 1 film.

1985), which was found to be enriched approximately 2fold in antigen reactive with mAb 44D 10 compared with the homogenate (Table 1). Most of the antigen in the P3 fraction was recovered in the Triton X-100 extract resulting in a 3.4-fold purification. The soluble extract was then applied sequentially to a non-immune IgG-Sepharose column and to an mAb 44D10IgG-Sepharose column. The enrichment in antigen in the bound and eluted fraction relative to the Triton X100 extract was of the order of 1000-fold, with a yield of 91 % of the total antigen present in the white matter homogenate (Table 1). The immunoadsorbed protein fraction had been enriched in 44D10 antigen approx. 4300-fold relative to the starting white-matter homogenate. Fig. 2 shows the bound and eluted protein fraction radiolabelled with 125I, fractionated by 10 % (w/v) SDS/PAGE and visualized by autoradiography. The predominant band migrates with an apparent Mr of 80000 (lane 1). When the bound and eluted material was immunoprecipitated, only the Mr-80000-band remains (lane 2). For preparative purposes the immunoabsorbed

Trpt 3.9 Cysteinyl residues were quantified by reduction of P-80 and radioalkylation with 14C-labelled iodoacetamide (see the Materials and methods section). The fraction corresponding to carboxymethylcysteine was collected and counted for radioactivity. From the specific activity of the [14C]-labelled iodoacetamide it was determined that there was a negligible amount of cysteine in P-80 (0.04 residues/100 residues). t Tryptophanyl residues were quantified by hydrolysis of P-80 glycoprotein in methanesulphonic acid followed by amino acid analysis. *

protein was dialysed, concentrated and subjected to preparative SDS/PAGE. The band corresponding to the P-80 glycoprotein was electroetuted and the purified glycoprotein was precipitated as described in the Materials and methods section. Amino acid composition The amino acid composition of P-80 (Table 2) was determined in eight separate hydrolyses on four independently purified batches of P-80. The composition was high in acidic and neutral amino acids and low in basic residues. A noteworthy observation is the absence of detectable levels of cysteinyl residues. Since the decreased migration of P-80 on reducing gels suggested the reduction of a disulphide bond, we explored the possibility that P-80 could contain a single cystine bond, which would be expected to yield about 0.25 residues per 100 residues on amino acid analysis. To quantify the amount of cysteine, a small sample of glycoprotein was reduced with ,-mercaptoethanol and radioalkylated with "C-labelled iodoacetamide as described in the Materials and methods section. The reduced and alkylated glycoprotein was hydrolysed and derivatized as described. The area on the chromatogram which is known to contain carboxymethylcysteine was collected and the radioactivity measured. The specific activity of this peak corresponds to approximately 0.33 mol of 19g8-

Human brain P-80 glycoprotein Table 3. Carbohydrate analyses of purified P-80 glycoprotein

10-3x Mr

*:,^i-o._

.r*:'S;,}i.>eg°'.:

. _.S-

1

2

.

....

355

:._}::

Residues/ 100 Sugar analysis

G.l.c.

.:

Dionex

::: .:.

' 8' a =>

*

Amino sugars (n) Glucosamine (4) Galactosamine (3) Neutral sugars Fucose Galactose Mannose Sialic acid From amino acid analyses

3.7 + 1.0* 1.7 + 0.2

..

3.5t

..

.:

1.2

.

...

:.

.: .:

.: ..... S. .:

:.

.....

::....

::...::: .:

1.5 1.9t 8.5 5.5 4.2 3.2 3.2§ after 5 h hydrolysis in 4.0 M-

:.

:::

..

94 > ^ ^s

_ ..: 67e Fs a > ;,..

.X_... _: sifi

..8.._R..

*:

HCI.

.. .-e i.-.. i.. :..Y. .:.i:}. :::

t Determined by anion-exchange pulse-amperometric de-

::i::.

tection without derivatization. $ Gas chromatographic analysis after derivation. §- Determined by the method of Aminoff (1961).

::. ::

::....:.:

.::

:::.

.::. :.:

*:. ..::.

:: :. ::.... .: .: :.

:::

.: ....

::

carboxymethylcysteinyl residues per mol of polypeptide, i.e. 0.04 residues per 100. We concluded that P-80 did not contain cystine, as determined by these methods. Carbohydrate analyses Amino sugar analyses (glucosamine and galactosamine) of the purified P-80 preparations from brain were done by amino acid analyses and analyses of fucose, galactose and mannose by g.l.c. These results were in good agreement with those obtained in the Dionex' anion-exchange system. These results are summarized in Table 3. Amino sugar analyses reveal the presence of both glucosamine and galactosamine suggesting the presence of N-acetylglucosamine and Nacetylgalactosamine and, therefore, the presence of both N-linked and 0-linked oligosaccharides in the glycoprotein. Neutral sugars were analysed by g.l.c. as the trifluoroacetate derivatives of their 0methylglycosides. Analysis by this method showed that the P-80 glycoprotein contained fucose, galactose and mannose. Orosomucoid and bovine serum albumin were used as positive and negative standards respectively. Analyses by anion-exchange chromatography and detection by amperometric detection without derivatization were in good agreement with these data except that galactose was lower than obtained by g.l.c. A modification of the method of Aminoff (1961) was used to quantify the amount of sialic acid on the P-S0 glycoprotein (see the Materials and methods section). Using this method, we determined that P-80 contained 3.2 mol % sialic acid, or approx. 19 residues of sialic acid for each glycoprotein. Chemical deglycosylation of P-80 with TFMS TFMS, which cleaves N-linked oligosaccharides between the two N-acetylglucosamine residues and cleaves 0-linked oligosaccharides on the reducing end of the first N-acetylgalactosamine residue, was used to determine how much of the glycoprotein could be attributed to carbohydrate. After treatment of P-80 with TFMS, it was compared with the untreated P-80 on SDS/PAGE (Fig. I). TFMS deg-ycosylations eulted in Vol. 256

30Qo

Fig. 3. Chemical deglycosylation of P-80 glycoprotein with TFMS Gel-purified P-80 was radioiodinated using Iodogen and reacted with TFMS as described in the Materials and methods section. Samples before treatment (5000 c.p.m.) and after treatment (5000 c.p.m.) were fractionated by SDS/PAGE (10 %, w/v). Autoradiography was performed using XAR-1 film. Lane 1, before TFMS; lane 2, after TFMS. a shift in apparent Mr from 80000 to 55000, indicating that approximately 25000 Da of the glycoprotein consisted of carbohydrate.

DISCUSSION In the present study, we report the purification and partial characterization of an antigen (P-80) from human brain, which is reactive with mAb 44D10. A particulate fraction (P3)-, depleted of highly compacted myelin, was prepared from human white matter and the detergentsoluble extract of this particulate fraction was further purified by immunoaffinity chromatography, resulting in a 4300-fold purification of the antigen. The antigen was identified as a single polypeptide of Mr 80000. Amino sugar and neutral sugar analyses of the purified antigen indicated that it was a glycoprotein containing both Nand 0-linked oligosaccharides. Chemical cleavage of the oligosaccharides by TFMS followed by SDS/PAGE yielded a deglycosylated polypeptide of Mr 55 000, suggesting that carbohydrate constituted approx. 25000 of the native P-80 glycoprotein.

356

The apparent Mr of the antigen reactive with mAb 44D10 on lymphocytes has been reported as 85000 (Quackenbush et al., 1985), while the Mr of P-80 from several cultured cells was found to vary from 83000 to 87000 (N. Girgrah, M. Letarte & M. A. Moscarello, unpublished work). It has yet to be determined if the differences in Mr of P-80 glycoprotein prepared from brain, from lymphocytes and from cultured cells are due to variations in the carbohydrate composition, or in the length of the polypeptide backbone, or some combination of both. Variations in the nature of the oligosaccharides or the sites of attachment would suggest that the antigens from brain and lymphocytes represent glycoforms, as was demonstrated for Thy-I (Parekh et al., 1987). Three other monoclonal antibodies, 50B4, 50C7, and 50E6, generated against glycoproteins prepared from a detergent extract of chronic lymphocytic leukaemic cells, have been shown to immunoprecipitate homologous glycoproteins from brain (Mr 80000), from chronic lymphocytic leukaemic cells (Mr 85000) and from B lymphoblastoid cells (Letarte et al., 1985). The P-80 glycoprotein defined by this family of monoclonal antibodies was shown to be distributed over a wide range of leukaemic cells, cultured cell lines, and normal and malignant tissues (Letarte et al., 1985). Competitive inhibition studies (Letarte et al., 1985) showed that the P-80 glycoprotein was homologous to the antigen reactive with the F10.44.2 mAb originally described in brain, and on human T lymphocytes and granulocytes (Dalchau et al., 1980; McKenzie et al., 1982). The antigens reactive with mAb A1G3 (Haynes et al., 1983) or mAb A3D8 (Telen et al., 1983, 1984, 1985) were immunologically homologous to the P-80 glycoprotein defined in brain and lymphocytes by mAb 44D10 (Letarte et al., 1985). This large family of P-80 glycoprotein monoclonal antibodies were all shown to react with one of three spacially distinct epitopes on the glycoprotein (Letarte et al., 1985). Although these mAbs failed to react with tissues of other species, the phagocyte glycoprotein-1 series has been shown to have broad tissue and species reactivity (Isacke et al., 1986). Immunoperoxidase studies appear to have localized the P-80 glycoprotein to fibrous astrocytes in normal white matter (N. Girgrah, M. Letarte & M. A. Moscarello, unpublished work). It has yet to be determined whether the increased expression of P-80 in neurological diseases can be accounted for by proliferation of astrocytes as a response to tissue damage, or the induced expression of the glycoprotein on other cell types, both in white and grey matter, during the course of the disease. This work was supported by grants from the Medical Research Council of Canada (M.A.M.) and from the National Cancer Institute of Canada (M.L.) A postdoctoral fellowship

N. Girgrah and others (T.F.C.) and studentship (N.G.) were provided by the Multiple Sclerosis Society of Canada. M. L. is a Terry Fox scientist of the National Cancer Institute of Canada.

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Received 23 May 1988/18 July 1988; accepted 19 July 1988

1988