120
BlOCHEMlCAL SOCIETY TRANSACTIONS
Structure of the Complex Carbohydrate Chains in the Membrane Glycoproteinsof Semliki Forest Virus MAMA PESONEN and OSSI RJZNKONEN Department of Biochemistry, University of Helsinki, Haartmaninkatu 3 , 00290 Helsinki, Finland
The membrane proteins E l , E2 and E3 of Semliki Forest virus carry one complex carbohydrate chain each, consisting of sialic acid, galactose, N-acetylglucosamine, mannose and fucose (Pesonen & Renkonen, 1976). These chains were sequenced by stepwise degradation with exoglycosidases. Semliki-Forest virus grown in BHK cells was labelled in vivo with D-[l-14c]ghICOSamine hydrochloride (59mCi/mmol) or with ~-[l-~H]fucose (3.3Ci/mmol) (M. Pesonen & 0. Renkonen, unpublished work). The membrane proteins of the purified virus were isolated by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis (Matt ila et aZ., 1976). The polypeptides were digested with Pronase (Calbiochem). The glycopeptides were isolated by gel filtration and subjected to stepwise digestion with exoglycosidasesin the following order: a-D-neuraminidase (EC 3.2.1.1 8), B-D-galactosidase(EC 3.2.1.23), N-acetyl-B-D-hexosaminidase (EC 3.2.1.52), a-D-mannosidase (EC 3.2.1.24) Containing 8-D-mannosidase, again N-acetyl-8-D-hexosaminidase,a-L-fucosidase (EC 3.2.1.51) and finally N-acetyl-B-D-glucosaminidase(EC 3.2.1.50) (M. Pesonen & 0. Renkonen, unpublished work). Neuraminidase was purchased from Behringwerke; galactosidase and N-acetylhexosaminidase were gifts from Dr. Li, University of Tulane, New Orleans, LA, U.S.A.; the rest of the enzymes were obtained from Boehringer. After every enzyme digestion a sample was taken for gel filtration on Bio-Gel P-6 (BioRad) (Pesonen & Renkonen, 1976). The results are compatible with structures shown in Fig. 1. The details in the branching parts of the molecules, however, remain unknown. All complex carbohydrate chains are similar in their overall structure. However, the chain in E3 is larger than the others. In all chains the distal mannose units are a- and the proximal one is 8-anomeric (Pesonen & Renkonen, 1976). There are more sialic acid residues than galactose units in every chain. We do not know how these ‘extra’ units are bound. The structures presented are similar to those found in serum glycoproteins (Bayard & Roux, 1975). The same structures have tentatively been found on the surface of BHK cells (Ogata et al., 1976). Thus it seems that the host cell specifies the complex carbohydrate chains linked to the viral polypeptides.
(a)
(b)
AcNeu-Gal-G1c:NAc
\ AcNeu-Gal-GlcNAc -(Ma&--,/ GlcNAc
,
AcNeu-Gal-GlcNAc AcNeu-Gal-GlcNAc \ ‘(Man), AcNeu-Gal-GlcNAc’/ GlcNAc/
Pept i de
I I
GlcNAc-GlcNAc Fuc
Peptide
I
-(GlcNAc),-GlcNAc I Fk
Fig. 1. Structure of complexglycopeptide of membraneproteins El and E2 (a)and E3 (b) Abbreviations: GlcNAc, N-acetylglucosamine; Fuc, fucose; Gal, galactose; Man, mann9w; AcNeu, N-acetylneuraminic (sialic) acid. 1977
121
566th MEETING, CAMBRIDGE Bayard, B. & Roux, D. (1975) FEBS Lett. 55,206-207 Mattila, K., Luukkonen, A. & Renkonen, 0. (1976) Biochim. Biophys Acta 419,435-444 Ogata, S . I., Muramatsu, T. & Kobata, A. (1976) Nature (London)259. 580-582 Pesonen, M. & Renkonen, 0. (1976) Biochim. Biophys. Acta 45,51&525
Membrane Saccharides of Rat Liver and Malignant-Cell Nuclei ROBERT W. STODDART* and MICHAEL R. PRICEt *Strangeways Research Laboratory, Worts’ Causeway, Cambridge CBI 4RN, U.K.,and tCancer Research CampaignLaboratories, Universityof Nottingham, UniversityPark, Nottingham NG7 2RD, U.K.
In previous investigations on membranes of rat liver and hepatic tumour nuclei, differences were not observed between the normal and malignant state with respect to parameters, such as buoyant densitiesand DNA/protein ratios of isolated membranes (price et al., 1972), and to ultrastructural features (e.g. diameters of the nuclear-porecomplex) as assessed by a variety of electron-microscopictechniques (Harris et al., 1974). In the present study, discrimination between normal and neoplastic nuclear membranes has been achieved by using purified plant lectins to characterize the profile of various saccharide residues associated with nuclear surfaces. For this purpose, two test systems were used, one involving determination of the agglutinabilityof isolated nuclei by lectins and the other being a comparative analysis of the nuclear binding of fluorescein-labelled lectins and also aprotinin, a lectin-like substance of animal origin with specificity for carboxylic acid-containing polysaccharides (Kiernan & Stoddart, 1973; Stoddart & Kiernan, 1973). Nuclei were isolated from normal Wistar rat liver and two transplanted rat tumours as previously described (Price et al., 1972). These tumours were a 4dimethylaminoazobenzene-induced hepatoma, designated hepatoma D23 and a 3-methylcholanthreneinduced fibrosarcoma, designated sarcoma Mc7. Briefly, the method of isolation involves sedimentation of nuclei through a 60% (w/w) sucrose solution, followed by extensive centrifugal washing and h a 1 resuspension in a buffer consisting of 1mMNaHCOs, 2m~-CaC1,, 2m~-MgC1,, 150m~-NaCl(pH1: 7.6). The following plant agglutininswere used €or the identificationof specificsugar residues in membrane oligosaccharides: concanavalin A (Sigma Chemical Co., London S.W.6, U.K.) for a-Dglucose- and a-D-mannose-like residues; soya-bean agglutinin [prepared by R. D. Collins, R. W. Stoddart & W. Jacobson (unpublished work)] for N-acetyl-D-galactosamine-like residues; Ricinus communis agglutinin [prepared by R. D. Collins, R. W. Stoddart & W. Jacobson (unpublished work)] for D-galactose-like residues. Aprotinin (supplied as Trasylol by Bayer Pharmaceuticals, Haywards Heath, Sussex, U.K.) was used for the identification of material containing sialyl groups (Stoddart & Kiernan, 1973). Fluorescein isothiocyanate derivatives were prepared by the method of Smith & Hollers (1970). Agglutination tests were performed essentially as described by Nicolson et al. (1972) with nuclei adjusted to 1x 10’4 x lO’/ml. Table 1. Fluorescence staining of normal liver and tumour nuclei Intensity of staining was scored on a qualitative scale from 0 to (no staining to intense staining), If: indicates a detectable, but weak, reaction. Intensity of staining of nuclei with fluorescein isothiocyanate derivatives of:
++++
Nuclei preparation Liver Hepatoma D23 Sarcoma Mc7 VOl. 5
R. communis aggiutinin
++ k
+
-
Concanavalin A
+++ + to +$+
++to++++
Soya-bean agglutinin ++totF++ to
++ ++++ +++
Aprotinin +ftt
+ ++
BlOCHEMlCAL SOCIETY TRANSACTIONS
Structure of the Complex Carbohydrate Chains in the Membrane Glycoproteinsof Semliki Forest Virus MAMA PESONEN and OSSI RJZNKONEN Department of Biochemistry, University of Helsinki, Haartmaninkatu 3 , 00290 Helsinki, Finland
The membrane proteins E l , E2 and E3 of Semliki Forest virus carry one complex carbohydrate chain each, consisting of sialic acid, galactose, N-acetylglucosamine, mannose and fucose (Pesonen & Renkonen, 1976). These chains were sequenced by stepwise degradation with exoglycosidases. Semliki-Forest virus grown in BHK cells was labelled in vivo with D-[l-14c]ghICOSamine hydrochloride (59mCi/mmol) or with ~-[l-~H]fucose (3.3Ci/mmol) (M. Pesonen & 0. Renkonen, unpublished work). The membrane proteins of the purified virus were isolated by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis (Matt ila et aZ., 1976). The polypeptides were digested with Pronase (Calbiochem). The glycopeptides were isolated by gel filtration and subjected to stepwise digestion with exoglycosidasesin the following order: a-D-neuraminidase (EC 3.2.1.1 8), B-D-galactosidase(EC 3.2.1.23), N-acetyl-B-D-hexosaminidase (EC 3.2.1.52), a-D-mannosidase (EC 3.2.1.24) Containing 8-D-mannosidase, again N-acetyl-8-D-hexosaminidase,a-L-fucosidase (EC 3.2.1.51) and finally N-acetyl-B-D-glucosaminidase(EC 3.2.1.50) (M. Pesonen & 0. Renkonen, unpublished work). Neuraminidase was purchased from Behringwerke; galactosidase and N-acetylhexosaminidase were gifts from Dr. Li, University of Tulane, New Orleans, LA, U.S.A.; the rest of the enzymes were obtained from Boehringer. After every enzyme digestion a sample was taken for gel filtration on Bio-Gel P-6 (BioRad) (Pesonen & Renkonen, 1976). The results are compatible with structures shown in Fig. 1. The details in the branching parts of the molecules, however, remain unknown. All complex carbohydrate chains are similar in their overall structure. However, the chain in E3 is larger than the others. In all chains the distal mannose units are a- and the proximal one is 8-anomeric (Pesonen & Renkonen, 1976). There are more sialic acid residues than galactose units in every chain. We do not know how these ‘extra’ units are bound. The structures presented are similar to those found in serum glycoproteins (Bayard & Roux, 1975). The same structures have tentatively been found on the surface of BHK cells (Ogata et al., 1976). Thus it seems that the host cell specifies the complex carbohydrate chains linked to the viral polypeptides.
(a)
(b)
AcNeu-Gal-G1c:NAc
\ AcNeu-Gal-GlcNAc -(Ma&--,/ GlcNAc
,
AcNeu-Gal-GlcNAc AcNeu-Gal-GlcNAc \ ‘(Man), AcNeu-Gal-GlcNAc’/ GlcNAc/
Pept i de
I I
GlcNAc-GlcNAc Fuc
Peptide
I
-(GlcNAc),-GlcNAc I Fk
Fig. 1. Structure of complexglycopeptide of membraneproteins El and E2 (a)and E3 (b) Abbreviations: GlcNAc, N-acetylglucosamine; Fuc, fucose; Gal, galactose; Man, mann9w; AcNeu, N-acetylneuraminic (sialic) acid. 1977
121
566th MEETING, CAMBRIDGE Bayard, B. & Roux, D. (1975) FEBS Lett. 55,206-207 Mattila, K., Luukkonen, A. & Renkonen, 0. (1976) Biochim. Biophys Acta 419,435-444 Ogata, S . I., Muramatsu, T. & Kobata, A. (1976) Nature (London)259. 580-582 Pesonen, M. & Renkonen, 0. (1976) Biochim. Biophys. Acta 45,51&525
Membrane Saccharides of Rat Liver and Malignant-Cell Nuclei ROBERT W. STODDART* and MICHAEL R. PRICEt *Strangeways Research Laboratory, Worts’ Causeway, Cambridge CBI 4RN, U.K.,and tCancer Research CampaignLaboratories, Universityof Nottingham, UniversityPark, Nottingham NG7 2RD, U.K.
In previous investigations on membranes of rat liver and hepatic tumour nuclei, differences were not observed between the normal and malignant state with respect to parameters, such as buoyant densitiesand DNA/protein ratios of isolated membranes (price et al., 1972), and to ultrastructural features (e.g. diameters of the nuclear-porecomplex) as assessed by a variety of electron-microscopictechniques (Harris et al., 1974). In the present study, discrimination between normal and neoplastic nuclear membranes has been achieved by using purified plant lectins to characterize the profile of various saccharide residues associated with nuclear surfaces. For this purpose, two test systems were used, one involving determination of the agglutinabilityof isolated nuclei by lectins and the other being a comparative analysis of the nuclear binding of fluorescein-labelled lectins and also aprotinin, a lectin-like substance of animal origin with specificity for carboxylic acid-containing polysaccharides (Kiernan & Stoddart, 1973; Stoddart & Kiernan, 1973). Nuclei were isolated from normal Wistar rat liver and two transplanted rat tumours as previously described (Price et al., 1972). These tumours were a 4dimethylaminoazobenzene-induced hepatoma, designated hepatoma D23 and a 3-methylcholanthreneinduced fibrosarcoma, designated sarcoma Mc7. Briefly, the method of isolation involves sedimentation of nuclei through a 60% (w/w) sucrose solution, followed by extensive centrifugal washing and h a 1 resuspension in a buffer consisting of 1mMNaHCOs, 2m~-CaC1,, 2m~-MgC1,, 150m~-NaCl(pH1: 7.6). The following plant agglutininswere used €or the identificationof specificsugar residues in membrane oligosaccharides: concanavalin A (Sigma Chemical Co., London S.W.6, U.K.) for a-Dglucose- and a-D-mannose-like residues; soya-bean agglutinin [prepared by R. D. Collins, R. W. Stoddart & W. Jacobson (unpublished work)] for N-acetyl-D-galactosamine-like residues; Ricinus communis agglutinin [prepared by R. D. Collins, R. W. Stoddart & W. Jacobson (unpublished work)] for D-galactose-like residues. Aprotinin (supplied as Trasylol by Bayer Pharmaceuticals, Haywards Heath, Sussex, U.K.) was used for the identification of material containing sialyl groups (Stoddart & Kiernan, 1973). Fluorescein isothiocyanate derivatives were prepared by the method of Smith & Hollers (1970). Agglutination tests were performed essentially as described by Nicolson et al. (1972) with nuclei adjusted to 1x 10’4 x lO’/ml. Table 1. Fluorescence staining of normal liver and tumour nuclei Intensity of staining was scored on a qualitative scale from 0 to (no staining to intense staining), If: indicates a detectable, but weak, reaction. Intensity of staining of nuclei with fluorescein isothiocyanate derivatives of:
++++
Nuclei preparation Liver Hepatoma D23 Sarcoma Mc7 VOl. 5
R. communis aggiutinin
++ k
+
-
Concanavalin A
+++ + to +$+
++to++++
Soya-bean agglutinin ++totF++ to
++ ++++ +++
Aprotinin +ftt
+ ++
