Structural studies of the M blood group determinant

N o t e s to the E d i t o r

considering the restriction made upon quantitative results, these values imply an OER (oxygen enhancement ratio) equal to --~1.8, which is in good agreement with measured values (1.5 to 2) of this ratio in biologically equivalent systems 6.

occur both during and after irradiation, it can be assumed that the formation of these centres may involve primary and secondary radicals.

Conclusions

References

The proposed correlation implies that the number of radicals transformed by interaction with oxygen shows a dependence on the partial pressure of oxygen comparable to that observed in the rate of oxygen-induced inactivation of proteins, as determined by biological methods. Despite the considerable difference between biological and model systems, the above results point to an important role for free radicals in the inactivation mechanism. In this sense, from a biological point of view, the radicals that 'disappear' by being transformed into non-radical species (perhaps non-paramagnetic peroxides) could be responsible for the chemical damage. They can therefore be considered as latent centres of damage. As the radical conversion process is observed to

Structural studies of the M blood group determinant Ron L. Batstone-Cunningham, Robert F_.Hardy and Kilian Dill* Department of Chemistry, Clemson University, Clemson, SC 29631, USA (Received 2 March 1983; revised 27 April 1983)

Structural studies of homozygous glycophorin A M were undertaken by monitorino the 13C methyl resonances of 13C reductivel y methylated glycophorin A M (contains five N~,N-[13C]dimethyl Lys residues, and the N-terminal N~,N-[13C]dimethyl Ser residue) in various forms of glycosylation. The results indicate that removal of the ~-o-NeuAc residues does not affect the structure about the N-terminal Ser residue. However, removal of the fifteen O-linked oligosaccharide units results in a structural effect about the N-terminal Ser residue. Partial methylation experiments performed on native glycophorin A M and deglycosylated glycophorin A M indicate that methylation of the lysine residue(s) may influence the structure about the N-terminal Ser residue, especially in the case of deolycosylated A M. Keywords: Spectroscopy; M blood group determinant; carbohydrate residues; glycophorin A

Introduction The family of glycophorins are transmembrane sialoglycoproteins (60Yo carbohydrate by weight) of the human erythrocyte membrane 1, which are responsible for the display of the MN 2 and the Ss 3'4 antigens. Glycophorin A Mis solely responsible for the display of the M antigen 2. Data obtained by Furthmayr imply that the display of the MN blood group determinants by glycophorin A MNis due solely to the amino acid sequence difference between * To whom correspondence should be addressed. 0141-8130/83/050314-04503.00 (D 1983 Butterworth & Co. (Publishers) Ltd

314

Int. J. Biol. Macromol., 1983, Vol 5, October

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Patten, F. and Gordy, W. Rad. Res. 1964, 22, 29 Drew, J. and Gordy, W. Rad. Res. 1963, 18, 552 Gordy, W. and Shields, H. Rad. Res. Suppl. 1959, 1,491 Aripova, D. F. Biophysics 1967, 12, 1131 Aripova, D. F. and Eidus, L. Kh. Biophysics 1971, 16, 150 Dertinguer, H. and Jung, H. 'Molecular Radiation Biology', Springer-Verlag, Berlin Alper, T. Rad. Res. 1956, 5, 573 Kiefer, J. in '4th Symposium on Microdosimetry', Italy, Euratom 1973, Vol. 1, p. 441 Hunt, J. W. and William, J. Rad. Res. 1964, 23, 26 Wyard, S. J. J. Sei. Instr. 1965, 42, 769 Katayama, W. and Gordy, W. J. Chem. Phys. 1961, 35, 117 Crippa, P. R., Urbinati, E. U. and Vecli, A. J. Phys. (E) 1971, 4, 1701 Rezk, H. and Johsen, R. Int. J. Rad. Biol. 1978, 34, 337 Hutchinson, J. and Watts, K. Rad. Res. 1961, 14, 803

homozygous glycophorins A M and A N at positions 1 (Ser/Leu) and 5 (Gly/Glu)2. Other workers, studying chemical modification of the glycoprotein and antisera response, have drawn similar conclusions 5. Other groups, however, have done work implicating either the a-DNeuAc 6 or the lysine ~'8 residues in the expression of the MN antigens. In previous work 9'~°, we showed that the structures of the 13C reductively methylated glycophorins A M and AN differ considerably at and/or near the N-terminal amino acid residue. Total ~3C reductive methylation modifies the lysine residues (to give N',N-[lSC]dimethyl Lys) and the N-terminal amino acid residue (to give either N " , N [13C]dimethy I Ser or Leu, respectively) of glycophorins A M and A N. The 13C methyl resonance of N ~ , N [13C]dimethyl Leu in reductively methylated glycophorin A TM shows normal spectral behaviour, a single peak titrating with a pK a = 7.4 (Ref. 9). In contrast, the spectrum of the reductively methylated glycophorin A Mis very complex. Two resonances are observed for the N ~ , N [13C]dimethyl Ser residue. One resonance is sharp and is relatively unaffected by pH, the other is broad and titrates 9. Our previous work has also shown that the degree of glycosylation and the degree of methylation of the lysine residues affect the spectral behaviour of the ~3C reductively methylated N-terminal amino acid residues in heterozygous glycophorin A MN(Refs 9 and 10). Therefore, we have decided to examine the effects of various degrees of (i) glycosylation and (ii) reductive methylation of the lysines on the structural peculiarity exhibited in the Nterminal region of 13C reductively methylated glyco p h o r i n A M.

Experimental Homozygous glycophorin A M was isolated and totally 13C reductively methylated as described 9'1 o. Totally 13C reductively methylated desialoglycophorin A M was prepared by the same method used to prepare 13C

Notes to the Editor reductively methylated heterozygous A MN~0 Totally 13C reductively methylated deglycosylated glycophorin A (deglycoglycophorin A) was prepared by one of two methods: (i) The ~3C reductively methylated desialoglycophorin A Mwas deglycosylated using the procedure of Lisowska et al.~2 that we have previously, successfully, used in removing the fifteen O-linked oligosaccharide chains from glycophorin ~3. (ii) Native glycophorin A Mwas deglycosylated and then 13C reductively methylated using the same procedure we used for 13C reductively methylating native glycophorin A M. After removal of the O-linked oligosaccharides, the protein was exhaustively dialysed against distilled water and then freeze-dried. Neutral sugar assays were performed by the method of Winzler ~4 in order to quantify the loss of the carbohydrate residues. 13C-N.m.r. spectra were obtained on a Jeol-FX90Q at 22.5 MHz as described previously 11. Chemical shifts are given relative to a trace of internal 1,4-dioxane (added only when chemical shifts were determined), whose chemical shift was taken to be 67.86 ppm downfield from Me4Si.

has 'free' motion about the N-terminal residue while the other state experiences 'hindered' motion. Our current results together with previous work ~° seem to favour the second case. One other important point should be made concerning Figure la. The results seem to indicate that reductive methylation of the lysine residues may influence one of the 'states' of glycophorin A M. Much controversy has existed in the literature concerning the influence the carbohydrate residues (especially ctD-NeuAc) may have on the structure of the M and N determinants displayed by glycophorin A M'N. In order to investigate the influence the carbohydrate residues may have on the N-terminal structure of glycophorin A M, and hence the M determinant, we have monitored the ~3C resonances of ~3C reductively methylated glycophorin A M as a function of the degree of glycosylation of this species. Figure 2 shows these results at various pH values. At these pH values we should be able to separate the various resonances associated with the N~,N [~3C]dimethylserine species 9 and the chemical shift of the N',N-[-~3C]dimethyllysine resonance should not be affected 9. In comparing the vertically and horizontally expanded

Results and discussion Figure la shows the proton-decoupled ~3C-n.m.r. spectra of native glycophorin A M which had been treated with increasing, but limited, quantities of ~3C formaldehyde. Since the formaldehyde will react with the neutral aminospecies only and the pK~ values of the N-terminal amino and lysine e-amino groups differ by as much as 2 pH units 9, it is expected that the N-terminal Ser will be methylated first 9. The spectra in Figure la show this point. The upper trace is a spectrum of native glycophorin A M which has been treated with two equivalents of ~3Cformaldehyde. The lower trace is a spectrum of fully 13C reductively methylated glycophorin A M. The middle traces show the reaction of partially methylated glycophorin A M with increasingly larger quantities of 13C enriched formaldehyde. The large resonance (at 44.1 ppm) in the lower trace results from the 10 methyl carbons of the five N';,N-[~3C]dimethyllysine residues I of 13C reductively methylated glycophorin A M(Ref. 9). The sharp resonance at 43.3 ppm and the upfield broad shoulder (at ~42.8 ppm) result from the N~,N-[-13C]dimethyl Ser residue of fully ~3C reductively methylated glycophorin A M (Refs 9 and 10). It should be obvious in Figure la that the N-terminal Ser residue is reductively methylated first and then the lysine residues. However, note that as the lysine residues are increasingly dimethylated the sharp resonance at ---42.8 ppm (see second trace in Figure la) only becomes a broad shoulder in the lower trace in Figure la. Heating the sample to ~80°C sharpens this resonance once again 9'~°, but it did not have the same integrated intensity as the resonance at 43.3 ppm observed for some other samples 9. These results have previously been interpreted in two ways: (i) A chemical shift non-equivalence exists between the two methyl groups of NLN-[13C]dimethyl Ser residue resulting in hindered rotation about the C~-N bond with one of the methyl resonances being broadened due to hindered N~-CH3 rotation. (ii) Two states exist for glycophorin A Mwhich affect the reductively methylated N-terminal Ser residue. One state

ri

i'l

a

b

I

l

I

I

i

I

I

45

40

55

50

45

40

55

ppm "from MeaSi

ppm from Me4Si

Figure I Proton-decoupled 13C_n.m.r. spectra of the partial J3C reductive methylation studies of native homozygous glycophorin AM (~ 1.2 mM in H20, 30°C) and deglycosylated glycophorin A (~ 1.5 mM in H20, 30°C). Spectra of methylated samples were taken at a sample pH of ~7.3 and typically required 15 000-60 000 accumulations. Time-domain data were collected in 8192 addresses, with a recycle time of 1 s. A digital broadening of 2.8 Hz was applied during the processing of the data. The peak at 34.5 ppm in the spectra of traces (a) and (b) represents monomethylated iysine. (a) Native homozygous glycophorin A; the n.m.r, traces in the figure relate to the following molar ratios of formaldehyde:glycophorin in the reductive methylation reaction: 2.0, 8.0, 14.0, 24.0, 74.0. (b) Deglycosylated homozygous glycophorin A; the n.m.r, traces in the figure relate to the following molar ratios of formaldehyde:glycophorin in the reductive methylation reaction: 2.0, 4.0, 8.0, 16.0, 75.0

Int. J. Biol. Macromol., 1983, Vol 5, October

315

Notes to the Editor

13C-MM deglyco

Z3C-MM desiolo

I~C-MM nol'ive

4.0

/

'/

S

a I

/

/

o

Figure 2 A portion of the aliphatic region of the protondecoupled 13C-n.m.r. spectra of 13C reductively methylated homozygous glycophorin AM, )3C reductively methylated homozygous desialoglycophorin AM, and ~3C reductively methylated homozygous deglycosylated glycophorin AMat pH values 4.0, 7.3, and 9.0, respectively. Time-domain data were accumulated in 8192 addresses with a recycle time of Is. A digital broadening of 3.0 Hz was applied to the data. All glycophorin AM samples were ~ 1.2 mM (in H20, at 30°C) and required 10 000-20 000 accumulations

versions of 13C reductively methylated glycophorin A M (compare with Figure la lower trace) with those of 13C reductively methylated desialoglycophorin A M (desialo) no large difference can be observed. This work is in agreement with our previous results on heterozygous desialoglycophorin A MN (Ref. 10). This result also indicates that if the N-terminal Ser residue of glycophorin A M is truly responsible for the display of the M determinant, removal of the ct-D-NeuAc residues does not affect its structure. The spectra of 13C reductively methylated deglycosylated (deglyco) glycophorin A M on the other hand show marked differences compared with 13C reductively methylated glycophorin A M and desialoglycophorin A M at the N-terminal Ser residue. We have previously shown that deglycosylation of glycophorin A does not produce 'gross' structural changes in the remaining glycoprotein 13. Therefore, the structural influence of almost total carbohydrate removal may be ascribed to the carbohydrate containing portion of the molecule, near the N-terminus 1. The ~3C reductively methylated deglycosylated glycophorin A M sample shown in Fioure 2 was made by 13C reductive methylation of a sample of deglycosylated glycophorin A M (see methods). If the sample is first 13C reductively methylated and then deglycosylated, no signals for the N',N-[ ~3C]dimethyl Ser residue are observed in the spectrum of the sample (see Figure 3). This phenomenon was observed for three different glycophorin A M samples, several glycophorin A MN samples, and also for a glycophorin A TMsample. Heating of the samples to 80°C did not produce any new noticeable peaks. In order to show that we had not lost the N-terminal N~,N [13C]dhnethyl Ser labels, we hydrolysed the above sample and found resonances attributable to N~,N [ 13C]dimethy 1Ser and N',N-[ 13C]dimethy 1Lys in a ratio

316

Int. J. Biol. Macromol., 1983, Vol 5, October

b

Figure 3 A portion of the aliphatic region of the protondecoupled 13C-n.m.r. spectra of various ~3C reductively methylated deglycosylated glycophorins A. (a) about 1 mM 13C reductively methylated deglycosylated glycophorin AM (prepared by first reductively methylating the native glycophorin AM sample and then deglycosylating) in H20, pH 7.3 (~ 30°C). The spectrum required 9590 accumulations (1 s recycle time) and a digital broadening of 3.0 Hz was applied during the data processing. (b) About 1.2 mM a3C reductively methylated deglycosylated glycophorin AM (prepared by ~3C reductively methylating deglycosylated glycophorin AM) in H20, pH 7.2 (~30°C). The spectrum required 17061 accumulations (1 s recycle time) and a 3.0 Hz digital broadening was applied during the processing of the data

of 1:5 in the neutralized hydrolysate. In order to investigate the structural properties of deglycosylated glycophorin A M we have performed a partial 13C reductive methylation on this species. These results are shown in Figure lb. Clearly 'structural' changes near the N-terminal N~,N-[ 13C]dimethyl Ser residue are observed as lysine residues of the deglycosylated glycophorin are progressively reductively methylated. These results suggest that for the deglycosylated species the lysine may also play a role in the structural determination of the N-terminal residue, although it would appear to be somewhat different than for the case of native glycophorin A M"

Conclusion We have shown that multiple structures may exist for glycophorin A M. Moreover, the structure about the N-

Notes to the Editor

terminal Ser residue is not affected by the removal of the ctD-NeuAc residues but a structural change is observed if all of the O-linked carbohydrate units are removed. We have also shown that reductive methylation of the lysine residues has an effect on the N-terminal structure of glycophorin A M. Acknowledgements

3 4 5 6 7 8 9

This work was supported by a Research Corporation Grant. We also thank the Carolina Blood Center for outdated blood.

10

References

13 14

1 2

11 12

Dahr, W., Bielen, W., Beyreuther, K. and Kriiger, J. HoppeSeyler's Z. Physiol. Chem. 1980, 361, 145 Dahr, W., Beyreuthen, K., Steinbach, H., Gielen, W. and Krtiger, J. Hoppe-Seylers's Z. Physiol. Chem. 1980, 361,895 Lisowski, E. and Duk, M. Eur. J. Biochem. 1975, 54, 469 Springer, G. F. and Desai, P. R. 3. Biol. Chem. 1982, 257, 2744 Dahr, W., Uhlenbruck, G. and Knott, H. 3. lmmunogenet. 1975, 2, 87 Ebert, W., Metz, J. and Roelcke, D. Eur. J. Biochem. 1972, 27,470 Hardy, R. E., Batstone-Cunningham, R. L. and Dill, K. Arch. Biochem. Biophys. 1983, 222, 222 Batstone-Cunningham, R. L., Hardy, R. E., Daman, M. E. and Dill, K. Biochim. Biophys. Acta 1983, in press Hardy, R. E. and Dill, K. FEBS Lett. 1982, 143, 327 Lisowska, E., Duk, M. and Dahr, W. Carbohydr. Res. 1980, 79, 103 Daman, M. E. and Dill, K. Carbohydr. Res. 1983, 111,205 Winzler, R. J. Methods Biochem. Anal. 1955, 2, 279

Marchesi, V. T. Semin. Hematol. 1979, 16, 3 Furthmayr, H. Nature 1978, 271,519

Int. J. Biol. Macromol., 1983, Vol 5, October

317