Hum. Genet. 35, 335 343 (1977) © by Springer-Verlag1977
Different N-Terminal Amino Acids in the MN-Glycoprotein from M M and NAT Erythrocytes W. Dahr, G. Uhlenbruck, E. JanBen, and R. Schmalisch Abteilung ftir lmmunbiologie der Medizinischen Universit~tsklinik, Kerpener Strage 15, D-5000 K/51n41, Federal Republic of Germany
Summary. The major human erythrocyte membrane (MN-) sialoglycoprotein was purified from MM, MN, and N N cells using detergent gel and ion exchange chromatography. N-terminal analyses with dansyl-chloride revealed serine in preparations from M M and leucine in those from N N erythrocytes, whereas glycoprotein isolated from M N cells contained both the above amino acids. These data strongly suggest that the above residues may represent the structural difference between the M and N antigens. Evidence was also obtained that the Ss-glycoprotein, which is associated with "N" activity, exhibits the same N-terminal amino acid (leucine) as the MN glycoprotein from N N cells.
Introduction The involvement of carbohydrate in the M N antigen receptor sites is well established. Recent investigations, however, have provided various lines of indirect evidence that the structural difference between these human blood group antigens is not a function of the carbohydrate, but resides in the amino acid sequence of the major human red cell membrane sialoglycoprotein (MN-SGP) (Lisowska and Duk, 1975; Lisowska et al., 1975a; Fujita and Cleve, 1975; Ebert et al., 1973; Dahr et al., 1975a and b, 1976a--c; Uhlenbruck et al., 1976). Studies with modified SGPs suggest that an amino group electrostatically interacting with sialic acid (NANA) of an alkali-labile oligosaccharide (Thomas and Winzler, 1969) forms an essential part of the MN receptor site as recognized by common antisera, and that carbohydrate represents only a non-specific part of the antigens. Direct support for the above concept has been obtained by the preparation of M- and N-specific rabbit antisera reacting with glycoproteins from which all carbohydrate had been split off (Lisowska, 1976). Lisowska and Morawiecki (1967) discovered that various amino group reagents destroy the MN antigens with a concomitant blocking of e-amino groups of lysine (Lys) and ~-amino groups of serine (Ser) and leucine (Leu) in crude SGP
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p r e p a r a t i o n s f r o m M M a n d N N red cells. S u b s e q u e n t l y no N - t e r m i n a l a m i n o acid could be f o u n d for the M N - S G P or the m a j o r N - t e r m i n a l tryptic glycopeptide therefrom ( T h o m a s a n d Winzler, 1970; J a c k s o n a n d S e a m a n , 1972; Javaid a n d Winzler, 1974). This supported the a s s u m p t i o n that the N - t e r m i n a l a m i n o acid of the g l y c o p r o t e i n is chemically blocked ( T h o m a s a n d Winzler, 1970; Winzler, 1972) a n d in consequence, that a Lys residue is involved in the M N a n t i g e n receptor site. More recently T a n n e r a n d Boxer (1972) as well as Marchesi a n d co-workers (Segrest et al., 1973; T o m i t a a n d Marchesi, 1975) reported the presence of variable a m o u n t s of N - t e r m i n a l Ser a n d Leu in isolated M N - S G P . The p r e p a r a t i o n , termed g l y c o p h o r i n by the latter group, was later s h o w n to represent a m i x t u r e of S G P s ( F u r t h m a y r et al., 1975). In a preceding paper ( D a h r et al., 1975b), we have suggested that the S e r / L e u heterogeneity detected by the above investigators might represent the structural difference b e t w e e n the M a n d N antigens. In the present c o m m u n i c a t i o n , we describe the results of N - t e r m i n a l analyses o n the isolated M N - S G P .
Materials Dansyl-chloride (1-dimethylaminonaphtalene-5-sulfonylchloride), amino acids, triton X-100 and dimethylformamide were obtained from Serva (Heidelberg). 2-monochloroethanol was purchased from Fluka (Buchs). Other solvents of analytical reagent grade or higher purity and silica gel G thin layer plates were products of Merck (Darmstadt). Membrane filters were bought from Millipore (Bedford). Sephadex G-200 (fine) and CM-sephadex C-50 were obtained from Pharmacia (Uppsala). Other materials have been described in preceding papers (Dahr et al., 1974, 1975b and c, 1976a, b and d; Uhlenbruck et al., 1976).
Methods Crude sialoglycoproteins (SGPs) were prepared by hot phenol/saline extraction (Uhlenbruck et al., 1968) from outdated blood transfusion units typed for MNSs antigens. Usually six units were pooled for each preparation. The crude substances were dissolved (10 mg/ml) in 0.1 M sodium-phosphate buffer(pH 7.0) containing 5% sodium-dodecylsulfate (SDS) and 0.25% dithioerythritol, incubated for 20 min at 50°C and subsequently applied to a column (dimensions 2.5 X 100 cm) of sephadex G-200 equilibrated and eluted with the above buffer containing 1% SDS. Fractions of four ml were collected at a flow rate of 12 ml/h. Tubes containing the MN-SGP were pooled and re-chromatographed on the above column after dialysis and freeze-drying. This fraction was dialized for two days against 40% ethanol or methanol and subsequently for three days against distilled water. After freeze-drying the substance was dissolved (5 mg/ml) in 0.02 M sodium-acetate buffer (pH 4.7) containing 0.5% triton, dialized against the same buffer for 12 h, and subsequently applied to a column (dimensions 2.5 X 30 cm) of CM-sephadex C-50 equilibrated and eluted with the above buffer at a flow rate of 20 ml/h. Fractions containing the MN-SGP, which was not bound to the column, were dialized against alcohol and water as described above. Sodium-chloride was added to give a concentration of 0.9%. The solution was re-extracted by the phenol/saline method and lyophilized after dialysis and membrane filtration. Fractions I and III from the sephadex G-200 column were recovered by lyophilization after dialysis against alcohol and water as described above. Standard dimethylaminonaphtalene-5-sulfonyl-(dansyl-) amino acids were prepared by the method of Gros and Labouesse (1969). O-dansyl-tyrosine and e-dansyl-Lys were obtained by
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treatment of c~-chymotrypsin with dansyl-chloride (dansylation) followed by acid hydrolysis. Dansylation of SGPs (20--30 nanomoles, calculated by assuming a molecular weight of 31000 and a NANA content of 20%) was performed according to Gros and Labouesse (1969) with some modifications. The incubation mixture contained 2 mg SDS instead of urea. The reaction was stopped after two hours by the addition of 1 ml acetone and 1 ml 0.1 N NaOH. After removal of dansyl-OH by dialysis, the solution was freeze-dried or evaporated to dryness, dissolved in a small amount of water, and an aliquot was withdrawn for an estimation of recovery by N A N A analysis (Warren, 1959). Alternatively, the method of Hartley (1970) was employed, using a twenty fold volume containing 1 mg SDS and a reaction time of two hours. Hydrolyses were performed in 6 N HC1 at l l0°C under nitrogen for four hours. Quantities corresponding to up to 10 nanomoles of SGP were separated by mono- or bidimensional thin layer chromatography (Gros and Labouesse, 1969), and the spots visualized by irradiation at 366 nm using an Uvis irradiator (Desaga, Heidelberg). Other methods have been described elsewhere (Dahr et. al., 1974, 1975c, 1976b and e; Uhlenbruck et al., 1976).
Results Gel filtration of crude S G P s in the presence of sodium-dodecylsulfate (SDS) resulted in the f r a c t i o n a t i o n shown in Figure 1, which is similar to that o b t a i n e d by F u r t h m a y r et al. (1975). The m a j o r (MN) S G P was present in fraction II. As expected f r o m previous results ( D a h r et al., 1976e), this glycoprotein gave rise to a single b a n d (PAS-1), when separated by S D S - p o l y a c r y l a m i d e gel electrophoresis ( S D S - P A G E ) according to W e b e r and O s b o r n (1969), whereas two b a n d s (PAS-1 a n d PAS-2) were demonstrable, when the material was r u n by d i s c o n t i n o u s (disc) S D S - P A G E (Laemmli, 1970), a n d the relative a m o u n t m i g r a t i n g as PAS-1 a n d P A S - 2 was f o u n d to be c o n c e n t r a t i o n d e p e n d e n t (Fig. 2 a - - f ) . The f o r m a t i o n of the m o n o m e r i c f o r m (PAS-2) of the M N - S G P is prevented by the high buffer's ionic strength of the former electrophoretic system (V.T. Marchesi, pers. c o m m u n , a n d u n p u b l , data). F r a c t i o n II exhibited u n c h a n g e d M N a n d weak Ss antigen activity, attrib u t a b l e to a variable c o n t a m i n a t i o n by c o m p o n e n t F (trimeric form of PAS-3)
FI
F~I
FIll
3.0
2.0
1.0
Vo
0
0 TUBE
NUMBER
Fig. 1. Gel filtration of crude SGPs in the presence of SDS. Fractions were monitored for absorbance at 280 (m ----m) and 220 (1~ I,) nm as well as NANA (Warren, 1959) (o ----o).For further details see Materials and Methods. F= Fraction
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Fig. 2 a-- g. Typical patterns after S DS-electrophoretic separation of SGP fractions, a 50/~g of F II in 0.05 ml, b and c 3 ~g of F II R in 0.1 and 0.013 ml respectively, d and e 12/.~gof F II R in 0.02 and 0.1 ml respectively, f and g. 50 ~tg of F II in 0.1 ml. a SDS-PAGE, b - - f disc SDS-PAGE. a - - f stained by the periodic acid/Schiff (PAS-) procedure, g stained by the PAS- and superstained by the Coomassie Blue (CB-) procedure. PAS-2, PAS-1, components G and H and the minor protein bands are marked with an arrow in b, c, f and g respectively. Gels were not all run and stained on the same occasion. Mobilities and staining intensities are therefore not directly comparable
a n d / o r c o m p o n e n t G (mol. weight 89000 daltons). R e - e l e c t r o p h o r e s i s of gel slices c o n t a i n i n g this b a n d gave rise to P A S - I , P A S - 2 , P A S - 3 , a n d c o m p o n e n t B. As j u d g e d f r o m the a b o v e a n d o t h e r d a t a ( D a h r et al., 1976a and d), it m a y be r e p r e s e n t e d b y an a g g r e g a t e o f P A S - 1 a n d P A S - 3 . The a b o v e c o n t a m i n a t i n g aggregates were r e m o v e d by r e - c h r o m a t o g r a p h y ( F II R). This m a t e r i a l still c o n t a i n e d two m i n o r p r o t e i n s (Fig. 2g). These impurities a n d the detergents were r e m o v e d by c h r o m a t o g r a p h y on C M - s e p h a d e x a n d s u b s e q u e n t p h e n o l e x t r a c t i o n to give the isolated M N - S G P (Fig. 3 b a n d c). W h e n large a m o u n t s o f the isolated g l y c o p r o t e i n were s e p a r a t e d by disc S D S P A G E two m i n o r c o m p o n e n t s d e s i g n a t e d H a n d ! (mol. weights 102000 and 130000 d a l t o n s respectively) were detectable. In o r d e r to d e m o n s t r a t e t h a t the a b o v e b a n d s represent i n t e r c o n v e r t i b l e forms of the same molecule, gel slices c o n t a i n i n g the b a n d s were r e - r u n by disc S D S - P A G E . R e - e l e c t r o p h o r e s i s o f P A S - 2 or P A S - 1 yielded the s a m e b a n d s a n d c o m p o n e n t H. Conversely, the l a t t e r b a n d gave rise to P A S - 1 a n d P A S - 2 . C o m p o n e n t I was c o m p l e t e l y t r a n s f o r m e d into P A S - 1 , P A S - 2 , a n d b a n d H. The b a n d s H a n d I may, therefore, be r e g a r d e d as the tri- a n d t e t r a m e r i c f o r m o f P A S - 2 . F r a c t i o n I, which c o n t a i n e d aggregates with a mol. weight higher t h a n P A S - I as the p r e d o m i n a n t c o m p o n e n t s , was n o t investigated further. F r a c t i o n I I I c o n t a i n e d the two m i n o r g l y c o p r o t e i n s , designated Ss- a n d D - S G P s ( D a h r et al., 1976e), as well as M N - S G P in v a r i a b l e p r o p o r t i o n as j u d g e d f r o m their P A S staining intensity after (disc) S D S - P A G E .
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Fig. 3a--g. Typical patterns obtained after disc SDS-electrophoretic separation of SGP fractions, a 150 [~g of crude SGPs in 0.2 ml, b and e 50 and 75 Exgof isolated MN-SGP in 0.1 and 0.15 ml respectively, d - - f represent re-electrophoreses of gel slices containing component H, I, PAS-1 and PAS-2 respectively. Gels (a, b, and d--g) were stained by the PAS-, (c) by the CBprocedure. Components H and I are marked with an arrow in b. For details concerning the interrelationship of the bands with a mol. weight lower than PAS-1 see Dahr et al., 1976e
Table 1. MNSs blood group activities of isolated MN-SGP, and fraction III SGPs from cells of blood type
Crude SGPs Crude SGPs Crude SGPs Isolated M N - S G P Isolated MN-SGP Isolated MN-SGP Fraction III Fraction Ill
MMSs NNSs MMS-s-b MMSs MN a NNSs MMSs NNSs
Inhibitory activity for anti-M (rabbit)
anti-N (rabbit)
anti-S (human)
anti-s (human)
0.3 ~ 0.2 0.2 0.3 ~ 1.0 n.t.
3.5 0.3 ~ O 0.3 0.2 1.0 0.7
0.3 0.5 ¢0 ~ ~i ~ 0.1 0. l
3.5 3.5
1.3 1.3
Data are presented as the minimal concentration (mg/ml) completely inhibiting four agglutinating doses. The results represent average values obtained with three different sera a Similar results were obtained with MN-SGP isolated from MNSs, MNSS, and MNss red cells b Data obtained with SGPs prepared from an MMS-s-U-- and an MMS-s-U+ individual, which represent a mixture of MN- and D-SGPs (Dahr et al., 1976d), are shown for comparison Abbreviations: n.t. not tested, ~ = no inhibition at 30 m g / m l
T h e M N S s b l o o d g r o u p a c t i v i t i e s o f t h e i s o l a t e d M N - S G P a n d f r a c t i o n III, p r e p a r e d f r o m r e d cells o f d i f f e r e n t b l o o d t y p e s , a r e s h o w n i n T a b l e 1. A s c a n b e seen, the MN-activity of the purified major SGP was not significantly different f r o m t h a t o f t h e s t a r t i n g m a t e r i a l , w h e r e a s n o i n h i b i t i o n o f a n t i - S o r -s w a s obtained at high concentrations. In the case of SGPs from M M erythrocytes the
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340 Table 2. N-terminal amino acids in isolated MN-SGP, crude SGPs, and fraction III SGPs from cells of blood type
Isolated MN-SGP Isolated MN-SGP Isolated MN-SGP Crude SGPs Crude SGPs Crude SGPs Crude SGPs Fraction III Fraction III Fraction III
MMSs MN a MNSs MM MN NN MMS-sMMSs MN a NNSs
N-terminal amino acid
(2) (3) (2) (7) (4) (4) (2) (1) (3) (1)
Serine
Leucine
++++ ++ -++++ ++ (+) ++++ +++ ++ (+)
-++ +---+ ++ +++ ++++ -++++ ++++ +-~+
Numbers in parentheses indicate the number of preparations investigated. The number of crosses indicates the fluorescence intensity of the dansylderivatives using the method of Gros and Labouesse (1969). - - = spot not detectable a Preparations from MNSS, MNSs, and MNss erythrocytes
w e a k N ("N", D a h r et al., 1975 b) a n t i g e n was s e p a r a t e d f r o m the M N - S G P a n d r e c o v e r e d in f r a c t i o n III. N - t e r m i n a l analyses on the isolated M N - S G P f r o m M M a n d N N cells using the d a n s y l a t i o n m e t h o d o f G r o s a n d L a b o u e s s e (1969) yielded spots which were identified as d a n s y l - S e r a n d - L e u respectively. P r e p a r a t i o n s f r o m M N red cells c o n t a i n e d b o t h the a b o v e d a n s y l - d e r i v a t i v e s with a p p r o x i m a t e l y equal fluorescence i n t e n s i t y (Table 2). A p p l i c a t i o n of the m e t h o d to desialized M N - S G P did n o t cause a significant c h a n g e in the strength o f the spots. W h e n d a n s y l a t i o n was carried o u t on native M N - S G P p r e p a r a t i o n s a c c o r d i n g to H a r t l e y (1970), only a r a t h e r s m a l l a m o u n t o f d a n s y l - S e r a n d / or -Leu c o u l d be detected. If, however, the r e a c t i o n was p e r f o r m e d on desialized M N - S G P f r o m M M or N N e r y t h r o c y t e s , c o n s i d e r a b l y s t r o n g e r spots w e r e obtained. P a r t i a l b l o c k i n g o f the c a r b o x y l g r o u p ( a b o u t 60%) of N A N A by t r e a t m e n t o r S G P f r o m M M cells with c a r b o d i i m i d e / g l y c i n e - m e t h y l e s t e r ( H o a r e a n d K o s h l a n d , 1967; U h l e n b r u c k et al., 1976) h a d a similar, albeit less p r o n o u n c e d , effect. These d a t a suggest t h a t the low reactivity o f the M N - S G P ' s N - t e r m i n a l a m i n o acid by the l a t t e r d a n s y l a t i o n m e t h o d is due to the f o r m a t i o n of an i n t e r n a l ion p a i r involving the c a r b o x y l g r o u p o f N A N A , which c o u l d alter its p K and nucleophilicity ( H a r t l e y , 1970), o r to sterical h i n d r a n c e by sialic acid. Thin layer c h r o m a t o g r a p h i c e x a m i n a t i o n of the w a t e r phases after ether e x t r a c t i o n revealed the presence of a s t r o n g s p o t c o r r e s p o n d i n g to e-dansyl-Lys a n d a r a t h e r faint one, identified as O - d a n s y l - t y r o s i n e . The fluorescence intensity of these spots was n o t significantly affected b y r e m o v a l of N A N A o r the use o f one o r the o t h e r dansylation method. A r e l a t i o n s h i p b e t w e e n the a m o u n t o f N - t e r m i n a l S e t a n d Leu a n d the M N b l o o d t y p e was also revealed b y analyses on several crude S G P substances, a l t h o u g h b o t h d a n s y l - d e r i v a t i v e s were d e t e c t a b l e in all substances f r o m c o m m o n
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cells. It is significant that seven preparations from normal M M erythrocytes contained large amounts of Ser and small quantities of Leu, whereas only Ser could be detected in two preparations from M M S - s - red cells. These substances were shown (Dahr et al., 1975d, 1976d) to represent a mixture of MN-SGP and component D devoid of Ss-glycoprotein. This observation strongly suggests that the Ss-SGP exhibits Leu as the N-terminal amino acid. This notion is further supported by the observation that dansyl-Leu represented the major spot detectable by analyses on fraction III obtained from preparations differing in their MNSs activity (Table 2). The N-terminal amino acid of the minor glycoprotein corresponding to component D might be Ser, as no other significant dansyl-derivative besides Leu was obtained in fraction III. (See Note added in proof.) Discussion
The above data demonstrate clearly that the Ser/Leu heterogeneity at the Nterminal position of the MN-SGP is caused by a polymorphism, as has been suggested by Segrest et al. (1973) as well as Tomita and Marchesi (1975) on the basis of work on the unfractionated glycoprotein mixture (Furthmayr et al., 1975), rather than being caused by impurities. Our results offer the possibility that this polymorphism represents the structural difference between the M and N antigens. The alkali-labile oligosaccharide linked to the second position of the peptide chain (see Tomita and Marchesi, 1975), could be the structure required for the binding of common antisera and the MN-specific lectins. Preceding studies have provided evidence that the amino group, which is part of the MN antigen site, is involved in an electrostatic interaction with the carboxyl group of NANA (Ebert et al., 1973; Dahr et al., 1975 a; Uhlenbruck et al., 1976; Lisowska and Duk, 1975). The data obtained with the dansylation method of Hartley (1970) suggest that the MN-SGP's N-terminal amino acid fulfills this requirement. Alternatively, it is apparent from the results of Tomita and Marchesi (1975) that the lysines in the molecule are three or more residues apart from hydroxyamino acids carrying alkali-labile oligosaccharides. This supports the notion that an internal ion pair between the above groups might be rather unstable. Various data (Hamaguchi and Cleve, 1972; Lisowska et al., 1975b; Dahr et al., 1975b and d, 1976b, d, and f) and those presented above support the concept that all common red cells--regardless of their MN blood type--contain an additional N antigen, designated "N" (Dahr et al., 1975 b), which is located on the Ss-SGP. The above evidence that this glycoprotein exhibits the same N-terminal amino acid as MN-SGP from N N cells (leucine) is relevant in this context. It remains to be elucidated, whether or not the N-terminal peptide sequence and oligosaceharide attachment sites of the Ss-glycoprotein are structurally similar and eventually homologous to those of the MN-SGP a hypothesis proposed previously (Dahr et al., 1975b). It should be emphasized that the present investigations cannot rule out the possibility that the Ser/Leu polymorphism is closely genetically linked with the MN blood type, which could depend on a polymorphism at another position of
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the peptide chain. A n o t h e r heterogeneity ( g l y c i n e / g l u t a m i c acid) has been detected at the fifth p o s i t i o n of the M N - S G P ( T o m i t a a n d Marchesi, 1975). Various e x p e r i m e n t s to solve the a b o v e p r o b l e m are in progress in our laboratory.
Ackowledgements. We thank Dr. P. D. Issitt, Dr. M. N. Metaxas, and Dr. M. Metaxas-Btihler for blood samples and Dr. R. Newman for advice during preparation of the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 68/D 1). A preliminary report of this work has been presented at the Tenth Congress of the International Academy of Legal Medicine and Social Medicine, Munich, September 12--18, 1976. The congress report is about to be printed in the Wiener Beitr~ige zur gerichtlichen Medizin.
References Dahr, W., Uhlenbruck, G., Bird, G. W. G.: Cryptic A-like receptor sites in human erythrocyte glycoproteins: proposed nature of Tn-antigen. Vox. Sang. (Basel) 27, 2 9 4 2 (1974) Dahr, W., Uhlenbruck, G., Bird, G. W. G.: Influence of free amino and carboxyl groups on the specificity of plant anti-N. Vox. Sang. (Basel) 28, 389--391 (1975a) Dahr, W., Uhlenbruck0 G., Knott, H.: Immunochemical aspects of the MNSs blood group system. J. Immunogenet. 2, 87--100 (1975b) Dahr, W., Uhlenbruck, G., Gunson, H. H., Van der Hart, M.: Molecular basis of Tnpolyagglutinability. Vox Sang. (Basel) 29, 36--50 (1975c) Dahr, W., Uhlenbruck, B., Issitt, P. D., Allen Jr., F. H.: SDS-polyacrylamidegel electrophoretic analysis of the membrane glyeoproteins from S-s-U- erythrocytes. J. Immunogenet. 2, 249--251 (1975d) Dahr, W., Uhlenbruck, G., Leikola, J., Wagstaff, W., Landfried, K.: Studies on the membrane glycoprotein defect of En(a-)erythrocytes. I. Biochemical aspects. J. Immunogenet. 3, 329 346 (1976a) Dahr, W., Uhlenbruck, G., Wagstaff, W., Leikola, J.: Studies on the membrane glycoprotein defect of En(a-) erythrocytes. II. MN antigenic properties of En(a-) erythrocytes. J. Immunogenet. 3, 383--393 (1976b) Dahr, W., Uhlenbruck, G., Knott, H.: The defect of M k erythrocytes as revealed by sodiumdodecylsulfate polyacrylamide gel electrophoresis. J. Immunogenet. (in press 1976c) Dahr, W., Issitt, P. D., Uhlenbruck, G.: New concepts of the MNSs blood group system. In: Human blood groups. J. F. Mohn, ed. Basel: Karger (in press) 1976d Dahr, W., Ublenbruck, G., Janssen, E., Sehmalisch, R.: Heterogeneity of human red cell membrane sialoglyeoproteins. Blut 32, 171--186 (1976e) Dahr, W., Uhlenbruck, G., Schmalisch, R., Janssen, E.: Ss-blood type associated PAS-staining polymorphism of glycoprotein three from human erythrocyte membranes. Hum. Genet. 32, 121 132 (19761) Ebert, W., Metz, J., Roelcke, D.: Modifications of N-acetylneuraminic acid and their influence on the antigen activity of erythrocyte glyeoproteins. Eur. J. Bioehem. 27, 4 7 0 4 7 2 (1973) Furthmayr, H., Tomita, M., Marchesi V. T.: Fractionation of the major sialoglycopeptides of the human red cell membrane.Biochem, biophys. Res. Commun. 65, 113 121 (1975) Fujita, S., Cleve, H.: Isolation and partial characterization of two minor glyeoproteins form human erythrocyte membranes. Biochem. biophys. Acta Amst. 278, 271 280 (1975) Gros, C., Labouesse, B.: Study of the dansylation reaction of amino acids, peptides and proteins. Eur. J. Bioehem. 7, 463--470 (1969) Hamaguchi, H., Cleve, H.: Solubilizationof human erythrocyte membranes and separation of the MN glyeoprotein from a glyeoprotein with I, S and A activity. Bioehim. biophys.Acta Amst. 278, 271--280 (1972) Hartley, B. S.: Strategy and tactics in protein chemistry. The first BDH lecture. Biochem. J. 119, 805 822 (1970)
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Hoare, D. G., Koshland, D. E.: A method for the quantitative modification and estimation of carboxylic groups in proteins J. biol. Chem. 242, 2447 2453 (1967) Jackson, L. J., Seaman, G. V. F.: Physicochemical properties of some glycopeptides released from human erythrocyte membranes by trypsin. Biochemistry 11, 4 4 4 9 (1972) Javaid, J. I., Winzler, R. J.: Association of glycoproteins with membranes. I. Isolation and molecular weight of the monomeric unit of the major glycoprotein from human erythrocytes. Biochemistry 13, 3635--3638 (1974) Laemmli, U. K.: Cleavage of structural protein during the assembly of the head of bacteriophage T 4. Nature 227, 680--685 (1970) Lisowska, E.: Immunological properties of M and N blood group antigens and products of their degradation. In: Human blood groups, edited by J. F. Mohn, ed. Basel: Karger (in press) (1976) Lisowska, E., Morawiecki, A.: The role of free amino groups in the blood group activity of M and N mueoids. Eur. J. Bioehem. 2, 237--241 (1967) Lisowska, E., Duk, M.: Modification of amino groups of human erythrocyte glycoproteins and the new concept on the structural basis of M and N blood group specificities. Eur. J. Biochem. 54, 469~475 (1975) Lisowska, E., Duk, M., Kordowicz, M.: Problem of the structural differentiation of M and N blood group antigens. Paper presented at the XIYth Congress of the International Society of Blood Transfusion, Helsinki, July 27--August 1, 1975a Lisowska, E., Dzierzkowa-Borodej, W., Seyfried, H., Drzeniek, Z.: Reactions of erythrocyte glycoproteins and their degradaion products with various anti-I sera. Vox Sang. (Basel) 28, 122--132 (1975b) Segrest, J. P., Kahane, I., Jackson, R. L., Marchesi, V. T.: Major glycoprotein of the human erythrocyte membrane: Evidence for an amphipathic molecular structure. Arch. Biochem. Biophys. 155, 167--183 (1973) Tanner, M. J. A., Boxer, D. H.J: Separation and some properties of the major proteins of the human erythrocyte membrane. Biochem. J. 129, 333 347 (1972) Thomas, D. B., Winzler, R. J.: Structural studies on human erythrocyte glycoproteins. Alkalilabile oligosaccharides. J. biol. Chem. 244, 5943 5946 (1969) Thomas, D. B., Winzler, R. J.: Glycoprotein antigens isolated from RBC, In: Blood and tissue antigens. D. Aminoff, ed. New York: Academic Press 1970 Tomita, M., Marchesi, V. T.: Amino acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc. Nat. Acad. Sci. 72, 2964--2968 (1975) Uhlenbruck, G., Rothe, A., Pardoe, G. I.: Bemerkenswerte chemische, serologische und elektrokinetische PNinomene bei Katzenerythroeyten: zugleich ein Beitrag zur Bestimmung und Bedeutung von mucoid- und glykolioidgebundener Erythrocytenneuramins/~ure. Z. Immunforsch. 136, 79 97 (1968) Uhlenbruck, G., Dahr, W., Schmalisch, R., Janssen, E.: Studies on the receptors of the MNSs blood group system.Blut 32, 163--170 (1976) Warren, L.: The thiobarbituric acid assay of sialic acids. J. biol. Chem. 234, 1971--1975 (1959) Weber, K., Osborn, M.: The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406--4412 (1969) Winzler, R. J.: Glycoproteins of plasma membranes. Chemistry and function. In: Glycoproteins, their composition, structure and function. A. Gottschalk, ed. Amsterdam: Elsevier 1972
Received September 21, 1976
Note A d d e d in P r o o f N-terminal analyses on SGPs prepared from blood of several single donors yielded the same results as obtained above with pooled material. Quantitative determination (Gros and Labouesse, 1969) revealed the Leu/Ser ratio to be higher for M M S S t h a n for M M s s SGPs, reflecting a relatively higher amount of Ss-glycoprotein previously detected by SDSPAGE methods (Dahr et al., 1976f).
Different N-Terminal Amino Acids in the MN-Glycoprotein from M M and NAT Erythrocytes W. Dahr, G. Uhlenbruck, E. JanBen, and R. Schmalisch Abteilung ftir lmmunbiologie der Medizinischen Universit~tsklinik, Kerpener Strage 15, D-5000 K/51n41, Federal Republic of Germany
Summary. The major human erythrocyte membrane (MN-) sialoglycoprotein was purified from MM, MN, and N N cells using detergent gel and ion exchange chromatography. N-terminal analyses with dansyl-chloride revealed serine in preparations from M M and leucine in those from N N erythrocytes, whereas glycoprotein isolated from M N cells contained both the above amino acids. These data strongly suggest that the above residues may represent the structural difference between the M and N antigens. Evidence was also obtained that the Ss-glycoprotein, which is associated with "N" activity, exhibits the same N-terminal amino acid (leucine) as the MN glycoprotein from N N cells.
Introduction The involvement of carbohydrate in the M N antigen receptor sites is well established. Recent investigations, however, have provided various lines of indirect evidence that the structural difference between these human blood group antigens is not a function of the carbohydrate, but resides in the amino acid sequence of the major human red cell membrane sialoglycoprotein (MN-SGP) (Lisowska and Duk, 1975; Lisowska et al., 1975a; Fujita and Cleve, 1975; Ebert et al., 1973; Dahr et al., 1975a and b, 1976a--c; Uhlenbruck et al., 1976). Studies with modified SGPs suggest that an amino group electrostatically interacting with sialic acid (NANA) of an alkali-labile oligosaccharide (Thomas and Winzler, 1969) forms an essential part of the MN receptor site as recognized by common antisera, and that carbohydrate represents only a non-specific part of the antigens. Direct support for the above concept has been obtained by the preparation of M- and N-specific rabbit antisera reacting with glycoproteins from which all carbohydrate had been split off (Lisowska, 1976). Lisowska and Morawiecki (1967) discovered that various amino group reagents destroy the MN antigens with a concomitant blocking of e-amino groups of lysine (Lys) and ~-amino groups of serine (Ser) and leucine (Leu) in crude SGP
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p r e p a r a t i o n s f r o m M M a n d N N red cells. S u b s e q u e n t l y no N - t e r m i n a l a m i n o acid could be f o u n d for the M N - S G P or the m a j o r N - t e r m i n a l tryptic glycopeptide therefrom ( T h o m a s a n d Winzler, 1970; J a c k s o n a n d S e a m a n , 1972; Javaid a n d Winzler, 1974). This supported the a s s u m p t i o n that the N - t e r m i n a l a m i n o acid of the g l y c o p r o t e i n is chemically blocked ( T h o m a s a n d Winzler, 1970; Winzler, 1972) a n d in consequence, that a Lys residue is involved in the M N a n t i g e n receptor site. More recently T a n n e r a n d Boxer (1972) as well as Marchesi a n d co-workers (Segrest et al., 1973; T o m i t a a n d Marchesi, 1975) reported the presence of variable a m o u n t s of N - t e r m i n a l Ser a n d Leu in isolated M N - S G P . The p r e p a r a t i o n , termed g l y c o p h o r i n by the latter group, was later s h o w n to represent a m i x t u r e of S G P s ( F u r t h m a y r et al., 1975). In a preceding paper ( D a h r et al., 1975b), we have suggested that the S e r / L e u heterogeneity detected by the above investigators might represent the structural difference b e t w e e n the M a n d N antigens. In the present c o m m u n i c a t i o n , we describe the results of N - t e r m i n a l analyses o n the isolated M N - S G P .
Materials Dansyl-chloride (1-dimethylaminonaphtalene-5-sulfonylchloride), amino acids, triton X-100 and dimethylformamide were obtained from Serva (Heidelberg). 2-monochloroethanol was purchased from Fluka (Buchs). Other solvents of analytical reagent grade or higher purity and silica gel G thin layer plates were products of Merck (Darmstadt). Membrane filters were bought from Millipore (Bedford). Sephadex G-200 (fine) and CM-sephadex C-50 were obtained from Pharmacia (Uppsala). Other materials have been described in preceding papers (Dahr et al., 1974, 1975b and c, 1976a, b and d; Uhlenbruck et al., 1976).
Methods Crude sialoglycoproteins (SGPs) were prepared by hot phenol/saline extraction (Uhlenbruck et al., 1968) from outdated blood transfusion units typed for MNSs antigens. Usually six units were pooled for each preparation. The crude substances were dissolved (10 mg/ml) in 0.1 M sodium-phosphate buffer(pH 7.0) containing 5% sodium-dodecylsulfate (SDS) and 0.25% dithioerythritol, incubated for 20 min at 50°C and subsequently applied to a column (dimensions 2.5 X 100 cm) of sephadex G-200 equilibrated and eluted with the above buffer containing 1% SDS. Fractions of four ml were collected at a flow rate of 12 ml/h. Tubes containing the MN-SGP were pooled and re-chromatographed on the above column after dialysis and freeze-drying. This fraction was dialized for two days against 40% ethanol or methanol and subsequently for three days against distilled water. After freeze-drying the substance was dissolved (5 mg/ml) in 0.02 M sodium-acetate buffer (pH 4.7) containing 0.5% triton, dialized against the same buffer for 12 h, and subsequently applied to a column (dimensions 2.5 X 30 cm) of CM-sephadex C-50 equilibrated and eluted with the above buffer at a flow rate of 20 ml/h. Fractions containing the MN-SGP, which was not bound to the column, were dialized against alcohol and water as described above. Sodium-chloride was added to give a concentration of 0.9%. The solution was re-extracted by the phenol/saline method and lyophilized after dialysis and membrane filtration. Fractions I and III from the sephadex G-200 column were recovered by lyophilization after dialysis against alcohol and water as described above. Standard dimethylaminonaphtalene-5-sulfonyl-(dansyl-) amino acids were prepared by the method of Gros and Labouesse (1969). O-dansyl-tyrosine and e-dansyl-Lys were obtained by
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treatment of c~-chymotrypsin with dansyl-chloride (dansylation) followed by acid hydrolysis. Dansylation of SGPs (20--30 nanomoles, calculated by assuming a molecular weight of 31000 and a NANA content of 20%) was performed according to Gros and Labouesse (1969) with some modifications. The incubation mixture contained 2 mg SDS instead of urea. The reaction was stopped after two hours by the addition of 1 ml acetone and 1 ml 0.1 N NaOH. After removal of dansyl-OH by dialysis, the solution was freeze-dried or evaporated to dryness, dissolved in a small amount of water, and an aliquot was withdrawn for an estimation of recovery by N A N A analysis (Warren, 1959). Alternatively, the method of Hartley (1970) was employed, using a twenty fold volume containing 1 mg SDS and a reaction time of two hours. Hydrolyses were performed in 6 N HC1 at l l0°C under nitrogen for four hours. Quantities corresponding to up to 10 nanomoles of SGP were separated by mono- or bidimensional thin layer chromatography (Gros and Labouesse, 1969), and the spots visualized by irradiation at 366 nm using an Uvis irradiator (Desaga, Heidelberg). Other methods have been described elsewhere (Dahr et. al., 1974, 1975c, 1976b and e; Uhlenbruck et al., 1976).
Results Gel filtration of crude S G P s in the presence of sodium-dodecylsulfate (SDS) resulted in the f r a c t i o n a t i o n shown in Figure 1, which is similar to that o b t a i n e d by F u r t h m a y r et al. (1975). The m a j o r (MN) S G P was present in fraction II. As expected f r o m previous results ( D a h r et al., 1976e), this glycoprotein gave rise to a single b a n d (PAS-1), when separated by S D S - p o l y a c r y l a m i d e gel electrophoresis ( S D S - P A G E ) according to W e b e r and O s b o r n (1969), whereas two b a n d s (PAS-1 a n d PAS-2) were demonstrable, when the material was r u n by d i s c o n t i n o u s (disc) S D S - P A G E (Laemmli, 1970), a n d the relative a m o u n t m i g r a t i n g as PAS-1 a n d P A S - 2 was f o u n d to be c o n c e n t r a t i o n d e p e n d e n t (Fig. 2 a - - f ) . The f o r m a t i o n of the m o n o m e r i c f o r m (PAS-2) of the M N - S G P is prevented by the high buffer's ionic strength of the former electrophoretic system (V.T. Marchesi, pers. c o m m u n , a n d u n p u b l , data). F r a c t i o n II exhibited u n c h a n g e d M N a n d weak Ss antigen activity, attrib u t a b l e to a variable c o n t a m i n a t i o n by c o m p o n e n t F (trimeric form of PAS-3)
FI
F~I
FIll
3.0
2.0
1.0
Vo
0
0 TUBE
NUMBER
Fig. 1. Gel filtration of crude SGPs in the presence of SDS. Fractions were monitored for absorbance at 280 (m ----m) and 220 (1~ I,) nm as well as NANA (Warren, 1959) (o ----o).For further details see Materials and Methods. F= Fraction
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Fig. 2 a-- g. Typical patterns after S DS-electrophoretic separation of SGP fractions, a 50/~g of F II in 0.05 ml, b and c 3 ~g of F II R in 0.1 and 0.013 ml respectively, d and e 12/.~gof F II R in 0.02 and 0.1 ml respectively, f and g. 50 ~tg of F II in 0.1 ml. a SDS-PAGE, b - - f disc SDS-PAGE. a - - f stained by the periodic acid/Schiff (PAS-) procedure, g stained by the PAS- and superstained by the Coomassie Blue (CB-) procedure. PAS-2, PAS-1, components G and H and the minor protein bands are marked with an arrow in b, c, f and g respectively. Gels were not all run and stained on the same occasion. Mobilities and staining intensities are therefore not directly comparable
a n d / o r c o m p o n e n t G (mol. weight 89000 daltons). R e - e l e c t r o p h o r e s i s of gel slices c o n t a i n i n g this b a n d gave rise to P A S - I , P A S - 2 , P A S - 3 , a n d c o m p o n e n t B. As j u d g e d f r o m the a b o v e a n d o t h e r d a t a ( D a h r et al., 1976a and d), it m a y be r e p r e s e n t e d b y an a g g r e g a t e o f P A S - 1 a n d P A S - 3 . The a b o v e c o n t a m i n a t i n g aggregates were r e m o v e d by r e - c h r o m a t o g r a p h y ( F II R). This m a t e r i a l still c o n t a i n e d two m i n o r p r o t e i n s (Fig. 2g). These impurities a n d the detergents were r e m o v e d by c h r o m a t o g r a p h y on C M - s e p h a d e x a n d s u b s e q u e n t p h e n o l e x t r a c t i o n to give the isolated M N - S G P (Fig. 3 b a n d c). W h e n large a m o u n t s o f the isolated g l y c o p r o t e i n were s e p a r a t e d by disc S D S P A G E two m i n o r c o m p o n e n t s d e s i g n a t e d H a n d ! (mol. weights 102000 and 130000 d a l t o n s respectively) were detectable. In o r d e r to d e m o n s t r a t e t h a t the a b o v e b a n d s represent i n t e r c o n v e r t i b l e forms of the same molecule, gel slices c o n t a i n i n g the b a n d s were r e - r u n by disc S D S - P A G E . R e - e l e c t r o p h o r e s i s o f P A S - 2 or P A S - 1 yielded the s a m e b a n d s a n d c o m p o n e n t H. Conversely, the l a t t e r b a n d gave rise to P A S - 1 a n d P A S - 2 . C o m p o n e n t I was c o m p l e t e l y t r a n s f o r m e d into P A S - 1 , P A S - 2 , a n d b a n d H. The b a n d s H a n d I may, therefore, be r e g a r d e d as the tri- a n d t e t r a m e r i c f o r m o f P A S - 2 . F r a c t i o n I, which c o n t a i n e d aggregates with a mol. weight higher t h a n P A S - I as the p r e d o m i n a n t c o m p o n e n t s , was n o t investigated further. F r a c t i o n I I I c o n t a i n e d the two m i n o r g l y c o p r o t e i n s , designated Ss- a n d D - S G P s ( D a h r et al., 1976e), as well as M N - S G P in v a r i a b l e p r o p o r t i o n as j u d g e d f r o m their P A S staining intensity after (disc) S D S - P A G E .
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Fig. 3a--g. Typical patterns obtained after disc SDS-electrophoretic separation of SGP fractions, a 150 [~g of crude SGPs in 0.2 ml, b and e 50 and 75 Exgof isolated MN-SGP in 0.1 and 0.15 ml respectively, d - - f represent re-electrophoreses of gel slices containing component H, I, PAS-1 and PAS-2 respectively. Gels (a, b, and d--g) were stained by the PAS-, (c) by the CBprocedure. Components H and I are marked with an arrow in b. For details concerning the interrelationship of the bands with a mol. weight lower than PAS-1 see Dahr et al., 1976e
Table 1. MNSs blood group activities of isolated MN-SGP, and fraction III SGPs from cells of blood type
Crude SGPs Crude SGPs Crude SGPs Isolated M N - S G P Isolated MN-SGP Isolated MN-SGP Fraction III Fraction Ill
MMSs NNSs MMS-s-b MMSs MN a NNSs MMSs NNSs
Inhibitory activity for anti-M (rabbit)
anti-N (rabbit)
anti-S (human)
anti-s (human)
0.3 ~ 0.2 0.2 0.3 ~ 1.0 n.t.
3.5 0.3 ~ O 0.3 0.2 1.0 0.7
0.3 0.5 ¢0 ~ ~i ~ 0.1 0. l
3.5 3.5
1.3 1.3
Data are presented as the minimal concentration (mg/ml) completely inhibiting four agglutinating doses. The results represent average values obtained with three different sera a Similar results were obtained with MN-SGP isolated from MNSs, MNSS, and MNss red cells b Data obtained with SGPs prepared from an MMS-s-U-- and an MMS-s-U+ individual, which represent a mixture of MN- and D-SGPs (Dahr et al., 1976d), are shown for comparison Abbreviations: n.t. not tested, ~ = no inhibition at 30 m g / m l
T h e M N S s b l o o d g r o u p a c t i v i t i e s o f t h e i s o l a t e d M N - S G P a n d f r a c t i o n III, p r e p a r e d f r o m r e d cells o f d i f f e r e n t b l o o d t y p e s , a r e s h o w n i n T a b l e 1. A s c a n b e seen, the MN-activity of the purified major SGP was not significantly different f r o m t h a t o f t h e s t a r t i n g m a t e r i a l , w h e r e a s n o i n h i b i t i o n o f a n t i - S o r -s w a s obtained at high concentrations. In the case of SGPs from M M erythrocytes the
W. Dahr et al.
340 Table 2. N-terminal amino acids in isolated MN-SGP, crude SGPs, and fraction III SGPs from cells of blood type
Isolated MN-SGP Isolated MN-SGP Isolated MN-SGP Crude SGPs Crude SGPs Crude SGPs Crude SGPs Fraction III Fraction III Fraction III
MMSs MN a MNSs MM MN NN MMS-sMMSs MN a NNSs
N-terminal amino acid
(2) (3) (2) (7) (4) (4) (2) (1) (3) (1)
Serine
Leucine
++++ ++ -++++ ++ (+) ++++ +++ ++ (+)
-++ +---+ ++ +++ ++++ -++++ ++++ +-~+
Numbers in parentheses indicate the number of preparations investigated. The number of crosses indicates the fluorescence intensity of the dansylderivatives using the method of Gros and Labouesse (1969). - - = spot not detectable a Preparations from MNSS, MNSs, and MNss erythrocytes
w e a k N ("N", D a h r et al., 1975 b) a n t i g e n was s e p a r a t e d f r o m the M N - S G P a n d r e c o v e r e d in f r a c t i o n III. N - t e r m i n a l analyses on the isolated M N - S G P f r o m M M a n d N N cells using the d a n s y l a t i o n m e t h o d o f G r o s a n d L a b o u e s s e (1969) yielded spots which were identified as d a n s y l - S e r a n d - L e u respectively. P r e p a r a t i o n s f r o m M N red cells c o n t a i n e d b o t h the a b o v e d a n s y l - d e r i v a t i v e s with a p p r o x i m a t e l y equal fluorescence i n t e n s i t y (Table 2). A p p l i c a t i o n of the m e t h o d to desialized M N - S G P did n o t cause a significant c h a n g e in the strength o f the spots. W h e n d a n s y l a t i o n was carried o u t on native M N - S G P p r e p a r a t i o n s a c c o r d i n g to H a r t l e y (1970), only a r a t h e r s m a l l a m o u n t o f d a n s y l - S e r a n d / or -Leu c o u l d be detected. If, however, the r e a c t i o n was p e r f o r m e d on desialized M N - S G P f r o m M M or N N e r y t h r o c y t e s , c o n s i d e r a b l y s t r o n g e r spots w e r e obtained. P a r t i a l b l o c k i n g o f the c a r b o x y l g r o u p ( a b o u t 60%) of N A N A by t r e a t m e n t o r S G P f r o m M M cells with c a r b o d i i m i d e / g l y c i n e - m e t h y l e s t e r ( H o a r e a n d K o s h l a n d , 1967; U h l e n b r u c k et al., 1976) h a d a similar, albeit less p r o n o u n c e d , effect. These d a t a suggest t h a t the low reactivity o f the M N - S G P ' s N - t e r m i n a l a m i n o acid by the l a t t e r d a n s y l a t i o n m e t h o d is due to the f o r m a t i o n of an i n t e r n a l ion p a i r involving the c a r b o x y l g r o u p o f N A N A , which c o u l d alter its p K and nucleophilicity ( H a r t l e y , 1970), o r to sterical h i n d r a n c e by sialic acid. Thin layer c h r o m a t o g r a p h i c e x a m i n a t i o n of the w a t e r phases after ether e x t r a c t i o n revealed the presence of a s t r o n g s p o t c o r r e s p o n d i n g to e-dansyl-Lys a n d a r a t h e r faint one, identified as O - d a n s y l - t y r o s i n e . The fluorescence intensity of these spots was n o t significantly affected b y r e m o v a l of N A N A o r the use o f one o r the o t h e r dansylation method. A r e l a t i o n s h i p b e t w e e n the a m o u n t o f N - t e r m i n a l S e t a n d Leu a n d the M N b l o o d t y p e was also revealed b y analyses on several crude S G P substances, a l t h o u g h b o t h d a n s y l - d e r i v a t i v e s were d e t e c t a b l e in all substances f r o m c o m m o n
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cells. It is significant that seven preparations from normal M M erythrocytes contained large amounts of Ser and small quantities of Leu, whereas only Ser could be detected in two preparations from M M S - s - red cells. These substances were shown (Dahr et al., 1975d, 1976d) to represent a mixture of MN-SGP and component D devoid of Ss-glycoprotein. This observation strongly suggests that the Ss-SGP exhibits Leu as the N-terminal amino acid. This notion is further supported by the observation that dansyl-Leu represented the major spot detectable by analyses on fraction III obtained from preparations differing in their MNSs activity (Table 2). The N-terminal amino acid of the minor glycoprotein corresponding to component D might be Ser, as no other significant dansyl-derivative besides Leu was obtained in fraction III. (See Note added in proof.) Discussion
The above data demonstrate clearly that the Ser/Leu heterogeneity at the Nterminal position of the MN-SGP is caused by a polymorphism, as has been suggested by Segrest et al. (1973) as well as Tomita and Marchesi (1975) on the basis of work on the unfractionated glycoprotein mixture (Furthmayr et al., 1975), rather than being caused by impurities. Our results offer the possibility that this polymorphism represents the structural difference between the M and N antigens. The alkali-labile oligosaccharide linked to the second position of the peptide chain (see Tomita and Marchesi, 1975), could be the structure required for the binding of common antisera and the MN-specific lectins. Preceding studies have provided evidence that the amino group, which is part of the MN antigen site, is involved in an electrostatic interaction with the carboxyl group of NANA (Ebert et al., 1973; Dahr et al., 1975 a; Uhlenbruck et al., 1976; Lisowska and Duk, 1975). The data obtained with the dansylation method of Hartley (1970) suggest that the MN-SGP's N-terminal amino acid fulfills this requirement. Alternatively, it is apparent from the results of Tomita and Marchesi (1975) that the lysines in the molecule are three or more residues apart from hydroxyamino acids carrying alkali-labile oligosaccharides. This supports the notion that an internal ion pair between the above groups might be rather unstable. Various data (Hamaguchi and Cleve, 1972; Lisowska et al., 1975b; Dahr et al., 1975b and d, 1976b, d, and f) and those presented above support the concept that all common red cells--regardless of their MN blood type--contain an additional N antigen, designated "N" (Dahr et al., 1975 b), which is located on the Ss-SGP. The above evidence that this glycoprotein exhibits the same N-terminal amino acid as MN-SGP from N N cells (leucine) is relevant in this context. It remains to be elucidated, whether or not the N-terminal peptide sequence and oligosaceharide attachment sites of the Ss-glycoprotein are structurally similar and eventually homologous to those of the MN-SGP a hypothesis proposed previously (Dahr et al., 1975b). It should be emphasized that the present investigations cannot rule out the possibility that the Ser/Leu polymorphism is closely genetically linked with the MN blood type, which could depend on a polymorphism at another position of
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the peptide chain. A n o t h e r heterogeneity ( g l y c i n e / g l u t a m i c acid) has been detected at the fifth p o s i t i o n of the M N - S G P ( T o m i t a a n d Marchesi, 1975). Various e x p e r i m e n t s to solve the a b o v e p r o b l e m are in progress in our laboratory.
Ackowledgements. We thank Dr. P. D. Issitt, Dr. M. N. Metaxas, and Dr. M. Metaxas-Btihler for blood samples and Dr. R. Newman for advice during preparation of the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 68/D 1). A preliminary report of this work has been presented at the Tenth Congress of the International Academy of Legal Medicine and Social Medicine, Munich, September 12--18, 1976. The congress report is about to be printed in the Wiener Beitr~ige zur gerichtlichen Medizin.
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Received September 21, 1976
Note A d d e d in P r o o f N-terminal analyses on SGPs prepared from blood of several single donors yielded the same results as obtained above with pooled material. Quantitative determination (Gros and Labouesse, 1969) revealed the Leu/Ser ratio to be higher for M M S S t h a n for M M s s SGPs, reflecting a relatively higher amount of Ss-glycoprotein previously detected by SDSPAGE methods (Dahr et al., 1976f).
