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Studies of human IgD myeloma proteins
Immunochemistry, 1977,
Vol. 14, pp. 393-396.
Pergamon Press.
Printed in Great Britain
STUDIES OF HUMAN IgD MYELOMA PROTEINS CIRCULAR DICHROISM OF INTACT PROTEIN SOME PROTEOLYTIC FRAGMENTS
AND
R. JEFFERIS, 1 J. B. M A T T H E W S 1 and P. M. BAYLEY 2 tDepartment of Experimental Pathology, University of Birmingham Medical School. Birmingham B15 2TJ, and 2National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7, U.K. (First received 9 November 1976; in revised form 27 January 1977)
Abstract--The C.D. spectra of two IgD myeloma proteins and their enzymatically derived Faba and F% fragments have been studied. The intact proteins differ from each other in the presence of a strong positive band at 235nm, present in one protein only. The structural feature responsible for this difference is clearly demonstrated to be localized within the Fab~ region. No significant conformational change is detected on proteolytic cleavage since the sum of the contributions of the fragments is equivalent to the intact protein.
INTRODUCTION
MATERIALS AND METHODS
Whilst a biological role for IgD has not been convincingly demonstrated, recent studies suggest that its primary function may be as an antigen receptor on the antigen sensitive small B lymphocyte (Rowe et al., 1973; Vitetta & Uhr, 1975). Biological activity has been demonstrated for several myeloma proteins including antigen binding (Swiercznska et aL, 1976) and the fixing of the complement component C3 by intact protein or F% fragments (Konno et al., 1975). The latter study demonstrates that C3 binding sites are revealed within Fc6 on cleavage of the Fab6 fragment from the intact molecule, but does not give information on whether this is due to the site being sterically blocked in the intact molecule or whether the C3 binding site is generated by a conformational change within the F% region following proteolysis in the hinge region. The generally observed sensitivity of IgD proteins to proteolysis has been interpreted as of possible biological significance with effector functions being revealed in the Fc6 region on removal of the Fab6 from membrane bound IgD (Vitetta & Uhr, 1975). The C.D. spectra of immunoglobulins have been shown to demonstrate characteristic features related to class and sub-class (Ghose, 1971; Ghose & Jirgensons, 1971a; Dorrington & Bennich, 1973; Johnson et al., 1974). Also, C.D. spectra can be used to delineate structural changes accompanying sub-unit interaction, the contribution of sub-units to overall conformation and conformational changes accompanying denaturation or proteolysis (Ghose & Jirgensons, 1971b; Cathou & Dorrington, 1975). In the present report we have employed C.D. to investigate structural relationships between lgD and the other immunoglobulin classes, to attempt to detect conformational change on proteolysis and to determine the sub-unit responsible for a positive absorption band at 235 nm observed in some IgD proteins.
Materials
Immunoglobulin D proteins were isolated from the sera of two patients (Ha and Ai) with multiple myeloma by the procedure of Jefferis (1976). Both proteins were shown to bear lambda chains by the use of specific antisera (Jefferis, 1975a). Enzyme digestions Trypsin digestion of IgD at 5 10 mg/ml was performed in 0.5% ammonium bicarbonate, pH 8.4, using 1"~ w/w trypsin at room temperature for two rain. Papain digestion was performed as described by Spiegelberg et al. (1970). Isolation of Fab~ and Fc~ fragments The tFab~ and tF% fragments were prepared from 2-rain digests with trypsin. The digest was dialyzed against 0.01 M phosphate buffer, pH 8.0, and applied to a column of Whatman DE-52 equilibrated and eluted with the same buffer. The tFab~ fragment is not absorbed under these conditions. The column was then washed with 0.02 M phosphate buffer, pH 8.0, and the tFco fragment eluted with phosphate buffered saline at pH 7.2. The papain Fc,~ fragment was prepared from a l-hr papain digest in the absence of cysteine as described by Spiegelberg et al, (1970). The tF% fragment of protein Ai was prepared by incubation of the papain F% fragment with trypsin for 2rain at room temperature and separated from the glycopeptide released by Sephadex G-100 gel filtration.
C.D. measurements Solutions for spectroscopy were routinely passed through Millipore filters (0.45 pore dia). Absorption spectra were measured on the Cary 118 recording Spectrophotometer, with samples in 1 cm optical cells. An extinction value of At@b,,m= 17.0 was used for both IgD and components. Circular dichroism was recorded on a Jouan Dichrographe liB fitted with a 150 W xenon arc. Solutions were at room temperature (20°C) in cells of path length l cm for the near u.v. measurements (350-250nmt and 1 mm for the far u.v. measurements (250-200nm). The absorbance of protein solutions was kept less than 1.0 at 280nm. For the far u.v., concentrations of the order of 393
IM\1 I 1 B A
394
R. JEFFERIS, J. B. MATTHEWS and P. M. BAYLEY
1] ~E
A
0f-
0.2 mg/ml were routinely used. The operating voltage was 560 760V for near u.v. and up to l l00V for far u.~. measurements. The experimental resuhs were recorded simultaneously on the chart recorder and in digital form via a BCD interface. Points were taken at 0.5-nm intervals, the spectra being scanned at 3.75 nm/min with a nominal instrumental time constant of 10sec. Multiple scanning of individual spectra was occasionally performed, but the spectra 104 reported here are those from single scans normalised to standard concentration by appropriate numerical factors. The C.D. data are presented in terms of AEM. the molar circular diehroism extinction coefficient, with units J '~/ ~cm ~. A%t is calculated from the observed C.D. 210 230 2,50 ' absorbancc AA by Aeu = (AA x 112/c × I}where c = con250 270 290 310 330 centration (mg/mlk I path length (m cm) and the factor 112 is the mean residue tool. wt [Bayley, 1973). Ae~a is Fig. 2. The fat (AI and near (BI u.~. C.D. spectra of the related to the molar residue ellipticity (0~t) with units deg tF% (O @), tFaba (Ik At and papain derived Fc,, c m 2 d m o l c by I")~t~= 3300 ,~ Ae u. (m i ) fragment of IgD protein Ha. Data processing invohed subtraction of instrumental baselines, normalising, smoothing by serial application of a 20-point second-order polynomial algorithm and plotting far u.v. spectra, Fig. 2A, whilst the Fab a fragment via Calcomp routines, These operations were programmed absorption is less intense. In the near u.v.C.D, both on the interactive graphics system (Taktronix 4010) in the Fca fragments are predominantly positive and show Hewlett Packard 3(X)0 Computing Laboratory of the the same features of detailed fine structure in the National Institute for Medical Research. region above 270 nm. Proteolytic fragments were also prepared from the lgD protein At. The Fab> and F'% fragments were isolated from a digest of IgDA~ with papain in the R ES t ! LTS absence of cysteine, and the tF% fragment obtained The C.D. spectra of lgD myeloma proteins Ha and by tryptic digestion of the larger papain F%. The C.D. Ai are shown in Fig. 1. In the far u.v. both proteins spectra are shown in Fig. 4. In the far u.v., Fig. 4A, show a negative trough at 215 217nm typical of im- the positive absorption feature exhibited by the intact munoglobulins. However, IgD,~ exhibits a strong protein IgDA~ is clearly shown to be contributed by positive band at 235 nm similar to that reported by a structural characteristic of the Fab,~ fragment. The Johnson et al. (1975). The near u.v. spectra of both spectra of the F% fragments are closely similar to proteins are characterized by bands which are posi- each other, being typical in form and somewhat tive overall showing aromatic fine structure above weaker than the Faba. In the near u.v., Fig, 4b, the 280nm and a broad positive absorption in the Fc,~ fragments again yield essentially identical spectra. 280--260nm region. The C.D. spectrum of an unfractionated 5-rain tryptic digest of IgD protein Ha was AA effectively identical to the spectrum of the intact protein, Thus, conformational changes resulting from 10 limited proteolysis were not detected. The C.D. spectra of the Faba and F% fragments obtained by digestion with trypsin (tFab a and tF%) and papain in the absence of cysteine are shown in Fig, 2. Both Fco fragments show similar and typical
'°i
20i
0
A ( x 1oo
B
A 30-
0.41AE
2010" 0-10-
0-0.4-_- / -0.8-
-5
2.5---r250
nm
nm
210
230
250
250
270
' 290
~
310
330
Fig. 1. The far (At and near (B) u.v.C.D, spectra of IgD myeloma proteins Ha (O Ot and Ai (A - At,
j 270
J--
~
290
~
~
310
nm
~ --1 330
Fig. 3. The near u.v.C.D spectra of lgDu~ t O - O} and the tFc,~ ( ~ - - - - - , ) and tFaba (kk) fragments derived lrom it. h~sct: the difference spectrum obtained between lgDj~, and the sum of the contributions of the tF% and tFaba fragments, scaled by 1/3 and 2,3, respectively. Units of AA are the experimental C.D. (absorbance units x lfls~ scaled to A 2 8 o = 1.0 for each protein.
C.D. Spectra of IgD Proteins and Fragments
395 DISCUSSION
The C.D. spectra of the intact IgD myeloma proteins studied exhibit an overall positive absorption in the 250-320 nm region in agreement with the data of Johnson et aL (1975). It is of interest to note that these features are more similar to those reported for IgE (Dorrington & Bennich, 1973) than for the other tO immunoglobulin classes (Johnson et al., 1973; Reisen et al., 1976). A feature of special interest is the positive band observed at 235 nm with protein Ai. The C.D. spectrum of an unfractionated tryptic digest of IgD protein Ha was indistinguishable from nm nm 210 230 250 250 270 290 310 3:30 that of the intact protein and thus we were unable to detect any conformational change resulting from Fig. 4. The far (A) and near (B) u.v.C.D, spectra of the cleavage in the hinge region. Similarly we show that tFc~ ( 0 - - - 0 ) , tFab~ (& A) and papain derived Fc~ the spectra of the isolated Fab~ and Fc~ fragments are additive and in sum equivalent to the intact pro(11--11) fragments of IgD protein Ai. tein (Fig. 3). It remains possible that biologically significant (Vitetta & Uhr, 1975; Konno et al., 1975) The Fab~ fragment is overall positive and clearly con- conformational changes may occur on cleavage in the tains the negative component at 300 nm seen in IgDAi hinge region that are not detectable on C.D. spectral and shows distinctive features at 292, 288 and 284 nm. analysis. Again reasonable additivity is shown for the scaled Comparison of the C.D. spectra of the Fc~ and Fc, sum of the F% and Fab6 components compared to (Dorrington & Bennich, 1973) fragments show that that of the intact protein IgDAi (Fig. 5). The deviation whilst Fc~ has positive bands at 295 nm and 288 nm here may be due to reliance on the absorption it goes negative at ca. 285 nm, and continues negative. measurements for concentration determination with However, it should be noted that the Fc, fragment scattering effects or the assumption of identical is composed of C,2, C,3 and C,4 domains and that absorption coefficients for all components leading to strong negative absorption bands in the 295-250 nm small differences. region are observed for the Fc" fragment which is In comparing the spectra of the Fab6 and Fc~ frag- essentially the isolated C,2 domain. This suggests that ments derived from IgD proteins Ha and Ai it is the C.D. spectrum of an Fc fragment composed of apparent that the spectra of the Fc6 are similar to the C,3 and C,4 domains might show positive features one another over the whole range 200-330 nm includ- similar to that observed for the Fc~ fragments. It is ing the detailed positions of the aromatic fine struc- the C,3 and C,4 domains which show the greatest ture in the region 280-300 nm. By contrast the Fab6 sequence homology with the F% fragment (Spiegelfragments are characteristically different with the berg, 1975). The studies of Ghose (1971) and Ghose Fab~ fragment derived from protein Ai exhibiting a and Jirgensons (1971a) have shown that F% has negapositive band at 235 nm, greater intensity at 270 nm tive absorption bands throughout its aromatic C.D. and a negative band at 300 nm. spectrum whilst the (Fc/t)5 fragment has positive absorption bands in the 300-280nm region which, interestingly, are shifted to lower wavelengths in the AA monomer Fc# fragment. However, the C.D. spectra of the F% fragments appear to be characteristic of 12 the IgD immunoglobulin class. The papain derived F% fragment differs from the tryptic in the presence of the hinge region of the delta chain. This hinge region has galactose and N-acetylgalactosamine present in strikingly similar proportions to that observed in lgA1 proteins (Baenzinger, 1974; Jefferis et aL, 1975b). However, whilst the polypeptide structure of the lgA1 protein contains an unusual duplicated sequence having a very high proline content (12 of 30 residues) we have found that the polypeptide of the hinge region of IgD proteins is of very different amino acid composition with a high basic amino acid content and only 3 of 30 residues to be proline (J. B. Matthews & R. Jefferis, unpubnm lished observation). No significant contribution of this 250 2to 290 hinge region structure to the C.D. absorption specFig. 5. The near u.v.C.D, spectra of IgDAi (0-----0) and trum of IgD proteins is observed since the spectra the scaled sum of the contributions of the tFca and tFab~ of the F% fragments derived from digestion with fragments (&___A) obtained from protein Ai. Units of papain or trypsin are essentially identical. AA are the experimental C.D. (absorbance units x l0 s) The C.D. spectra of the Fab6 fragments show posiscaled to A28o = 1.0 for IgDAi. tive bands in the 300-260 nm region broadly similar
396
R. JEFFERIS, J, B. MATTHEWS and P. M. BAYLEY
to those reported for Fab fragments derived from lgG, IgM and IgE (Ghose, 1971 : Ghose & Jirgensons, 1971; Dorrington & Bennich, 1973). The positive band at 235 nm observed with intact lgDx~ is present in the Fab0 fragment derived fiom il with increased intensity. The occurrence of an absorption band in this region for some immunoglobulin molecules has been reported in several studies (Cathou et al., 1968; Ghose & Jirgensons, 1971b; Johnson et al., 1974) and discussed in detail by Dorrington and Smith (1972), and Cathou and Dorrington {1975). It is thought to derive from one of the tyrosine residues present in the constant region of the kappa chain with the positive C.D. resulting from interaction between the variable and constant regions of kappa chains and is enhanced on interaction of the H and L chains. It has been stated (Cathou & Dorrington, 1975) that there is no good evidence that a comparable transition is detectable in htmbda chains. However, both IgD proteins under study in this report bear lambda chains (Jefferis, 1975a) and thus a comparable transition can be observed in some lgD proteins, at least, bearing lambda chains. Comparison of C~ and C,~ sequences (Dayhofl; 1973) reveal tyrosine residues at positions 146, 180, 193 and 199, and 145. 177 and 196, respectively. Obviously these tyrosine residues are present in very equivalent linear positions and it may be assumed that the conformation of C~ and C~. domains in intact proteins would be broadly similar, thus it would be expected that the interaction resulting in the positive C.D, band at 235nm for kappa chains might also be observed in molecules composed of lambda chains. We intend to take advantage of the possibility of isolating V.V,.C, and V.C~, fragments (Jefferis & Matthews, 1977; Jefferis, 1975a) to investigate further the interactions resulting in this C D . feature lbr lgD protein Ai. lckmm'le&tement This work was supported by a grant from the Medical Research Council.
NOTE ADDED IN PROOF
Since this paper was submitted the C.D. spectra o1 four further IgD proteins has been measured. They all gave spectra similar to that obtained with protein HA in this report; i.e. no other protein exhibited the positive absorption maxima at 235 nm.
REFERENCES
Baenzigcr ]. & Komfeld S. (1974) J. Biol. ('henl. 249. 7270 Bayley P. {19731 Pro q. Biophy~. moh'c. Biol. 27, 1. Cathou R. [!.. Kukzycki A. & Haber E, (19681 Bioche,tl>n,~ 7. 3958. Cathou R. E. & Dorrington K. ]. (1975) In 5ul)-tmii., i,~ Biological Systems, (Edited by Timesheff & Fasman~ Par1 C. Vol. 7. p. 154. Marcel Dekker, New York. Dayhoff M. O. (1973) Atlas ~!! Proteilt Sequem'c all,l S*rl,ture. Vol. 5, Suppl. I. National Biomedical Rcscalch Foundation, Silver Spring, MD. Oio E. & Jirgensons B. (1970) Bioctwmistrv 9, 1066. Dorrington K. J. & Smitla B. R. {[9721 Biochim. t~i,)?hl.< Actu 263, 70. Dorrington K. J. & Bennich H. ~1973)3. bir,I. ('he,i 248, 8378. Ghose A. ('. (19711 Biochem biophv.< Rc~. (ommlm 45, 1144. Ghose A. C. & Jirgensons B. 11971al ,h'chs Biochem. l'}i,phys. 144, 384. Ghosc A. C. & Jirgensons t< (lt~TIhl Hiochim. hioldl.l'< h'I,I 251, 14. Jefferis R. (1975a) Immured. Commun. 4, 477. Jefferis R., Butwell A. J. & ('lamp J. R. il975b) ('lit1. c~(~ hmmm. 22, 311. Jefferis R. (1976)J. Immwu)l. Metil. 9. 231. Jefferis R. & Matthews J. B. (1977) lmmunochemistrv 14, 171. Johnson P. M., Scopes P. M., Frac% B. M. & ~,Vatkm~, ]. 11974) hnmunoloqy 27, 27. Johnson P. M., Howard A. & Scopes P M. (1975t f.l..t~S Lett. 49, 310. Konno T.. Hirai H. & lnai S. 11075) lmmlimwlwmi.strl 12, 773. Reisen W. F.. Huser H, & Sk\aril [:. II9761 I E B S l,~tl. 61, 243. Rowe D. S., Hug K., Forni L, & Pernis g. {1~)73t 3. c',? Med. 138, 965. Spiege[berg H. L., Prahl V. W. &Grcg H. M. 11970) Bi,,chemi.~lry 231, 279. Spiegelberg H. L. (1975) Nature 254, 723. Swiercznska Z., Wozniczko-Orlowska G. & Maldyk tt. {1976) 1mmunochemistr.v 13, 379. Viletla F. S. & Uhr J. W. (1975) Sck,ncv 189, 964.
Vol. 14, pp. 393-396.
Pergamon Press.
Printed in Great Britain
STUDIES OF HUMAN IgD MYELOMA PROTEINS CIRCULAR DICHROISM OF INTACT PROTEIN SOME PROTEOLYTIC FRAGMENTS
AND
R. JEFFERIS, 1 J. B. M A T T H E W S 1 and P. M. BAYLEY 2 tDepartment of Experimental Pathology, University of Birmingham Medical School. Birmingham B15 2TJ, and 2National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7, U.K. (First received 9 November 1976; in revised form 27 January 1977)
Abstract--The C.D. spectra of two IgD myeloma proteins and their enzymatically derived Faba and F% fragments have been studied. The intact proteins differ from each other in the presence of a strong positive band at 235nm, present in one protein only. The structural feature responsible for this difference is clearly demonstrated to be localized within the Fab~ region. No significant conformational change is detected on proteolytic cleavage since the sum of the contributions of the fragments is equivalent to the intact protein.
INTRODUCTION
MATERIALS AND METHODS
Whilst a biological role for IgD has not been convincingly demonstrated, recent studies suggest that its primary function may be as an antigen receptor on the antigen sensitive small B lymphocyte (Rowe et al., 1973; Vitetta & Uhr, 1975). Biological activity has been demonstrated for several myeloma proteins including antigen binding (Swiercznska et aL, 1976) and the fixing of the complement component C3 by intact protein or F% fragments (Konno et al., 1975). The latter study demonstrates that C3 binding sites are revealed within Fc6 on cleavage of the Fab6 fragment from the intact molecule, but does not give information on whether this is due to the site being sterically blocked in the intact molecule or whether the C3 binding site is generated by a conformational change within the F% region following proteolysis in the hinge region. The generally observed sensitivity of IgD proteins to proteolysis has been interpreted as of possible biological significance with effector functions being revealed in the Fc6 region on removal of the Fab6 from membrane bound IgD (Vitetta & Uhr, 1975). The C.D. spectra of immunoglobulins have been shown to demonstrate characteristic features related to class and sub-class (Ghose, 1971; Ghose & Jirgensons, 1971a; Dorrington & Bennich, 1973; Johnson et al., 1974). Also, C.D. spectra can be used to delineate structural changes accompanying sub-unit interaction, the contribution of sub-units to overall conformation and conformational changes accompanying denaturation or proteolysis (Ghose & Jirgensons, 1971b; Cathou & Dorrington, 1975). In the present report we have employed C.D. to investigate structural relationships between lgD and the other immunoglobulin classes, to attempt to detect conformational change on proteolysis and to determine the sub-unit responsible for a positive absorption band at 235 nm observed in some IgD proteins.
Materials
Immunoglobulin D proteins were isolated from the sera of two patients (Ha and Ai) with multiple myeloma by the procedure of Jefferis (1976). Both proteins were shown to bear lambda chains by the use of specific antisera (Jefferis, 1975a). Enzyme digestions Trypsin digestion of IgD at 5 10 mg/ml was performed in 0.5% ammonium bicarbonate, pH 8.4, using 1"~ w/w trypsin at room temperature for two rain. Papain digestion was performed as described by Spiegelberg et al. (1970). Isolation of Fab~ and Fc~ fragments The tFab~ and tF% fragments were prepared from 2-rain digests with trypsin. The digest was dialyzed against 0.01 M phosphate buffer, pH 8.0, and applied to a column of Whatman DE-52 equilibrated and eluted with the same buffer. The tFab~ fragment is not absorbed under these conditions. The column was then washed with 0.02 M phosphate buffer, pH 8.0, and the tFco fragment eluted with phosphate buffered saline at pH 7.2. The papain Fc,~ fragment was prepared from a l-hr papain digest in the absence of cysteine as described by Spiegelberg et al, (1970). The tF% fragment of protein Ai was prepared by incubation of the papain F% fragment with trypsin for 2rain at room temperature and separated from the glycopeptide released by Sephadex G-100 gel filtration.
C.D. measurements Solutions for spectroscopy were routinely passed through Millipore filters (0.45 pore dia). Absorption spectra were measured on the Cary 118 recording Spectrophotometer, with samples in 1 cm optical cells. An extinction value of At@b,,m= 17.0 was used for both IgD and components. Circular dichroism was recorded on a Jouan Dichrographe liB fitted with a 150 W xenon arc. Solutions were at room temperature (20°C) in cells of path length l cm for the near u.v. measurements (350-250nmt and 1 mm for the far u.v. measurements (250-200nm). The absorbance of protein solutions was kept less than 1.0 at 280nm. For the far u.v., concentrations of the order of 393
IM\1 I 1 B A
394
R. JEFFERIS, J. B. MATTHEWS and P. M. BAYLEY
1] ~E
A
0f-
0.2 mg/ml were routinely used. The operating voltage was 560 760V for near u.v. and up to l l00V for far u.~. measurements. The experimental resuhs were recorded simultaneously on the chart recorder and in digital form via a BCD interface. Points were taken at 0.5-nm intervals, the spectra being scanned at 3.75 nm/min with a nominal instrumental time constant of 10sec. Multiple scanning of individual spectra was occasionally performed, but the spectra 104 reported here are those from single scans normalised to standard concentration by appropriate numerical factors. The C.D. data are presented in terms of AEM. the molar circular diehroism extinction coefficient, with units J '~/ ~cm ~. A%t is calculated from the observed C.D. 210 230 2,50 ' absorbancc AA by Aeu = (AA x 112/c × I}where c = con250 270 290 310 330 centration (mg/mlk I path length (m cm) and the factor 112 is the mean residue tool. wt [Bayley, 1973). Ae~a is Fig. 2. The fat (AI and near (BI u.~. C.D. spectra of the related to the molar residue ellipticity (0~t) with units deg tF% (O @), tFaba (Ik At and papain derived Fc,, c m 2 d m o l c by I")~t~= 3300 ,~ Ae u. (m i ) fragment of IgD protein Ha. Data processing invohed subtraction of instrumental baselines, normalising, smoothing by serial application of a 20-point second-order polynomial algorithm and plotting far u.v. spectra, Fig. 2A, whilst the Fab a fragment via Calcomp routines, These operations were programmed absorption is less intense. In the near u.v.C.D, both on the interactive graphics system (Taktronix 4010) in the Fca fragments are predominantly positive and show Hewlett Packard 3(X)0 Computing Laboratory of the the same features of detailed fine structure in the National Institute for Medical Research. region above 270 nm. Proteolytic fragments were also prepared from the lgD protein At. The Fab> and F'% fragments were isolated from a digest of IgDA~ with papain in the R ES t ! LTS absence of cysteine, and the tF% fragment obtained The C.D. spectra of lgD myeloma proteins Ha and by tryptic digestion of the larger papain F%. The C.D. Ai are shown in Fig. 1. In the far u.v. both proteins spectra are shown in Fig. 4. In the far u.v., Fig. 4A, show a negative trough at 215 217nm typical of im- the positive absorption feature exhibited by the intact munoglobulins. However, IgD,~ exhibits a strong protein IgDA~ is clearly shown to be contributed by positive band at 235 nm similar to that reported by a structural characteristic of the Fab,~ fragment. The Johnson et al. (1975). The near u.v. spectra of both spectra of the F% fragments are closely similar to proteins are characterized by bands which are posi- each other, being typical in form and somewhat tive overall showing aromatic fine structure above weaker than the Faba. In the near u.v., Fig, 4b, the 280nm and a broad positive absorption in the Fc,~ fragments again yield essentially identical spectra. 280--260nm region. The C.D. spectrum of an unfractionated 5-rain tryptic digest of IgD protein Ha was AA effectively identical to the spectrum of the intact protein, Thus, conformational changes resulting from 10 limited proteolysis were not detected. The C.D. spectra of the Faba and F% fragments obtained by digestion with trypsin (tFab a and tF%) and papain in the absence of cysteine are shown in Fig, 2. Both Fco fragments show similar and typical
'°i
20i
0
A ( x 1oo
B
A 30-
0.41AE
2010" 0-10-
0-0.4-_- / -0.8-
-5
2.5---r250
nm
nm
210
230
250
250
270
' 290
~
310
330
Fig. 1. The far (At and near (B) u.v.C.D, spectra of IgD myeloma proteins Ha (O Ot and Ai (A - At,
j 270
J--
~
290
~
~
310
nm
~ --1 330
Fig. 3. The near u.v.C.D spectra of lgDu~ t O - O} and the tFc,~ ( ~ - - - - - , ) and tFaba (kk) fragments derived lrom it. h~sct: the difference spectrum obtained between lgDj~, and the sum of the contributions of the tF% and tFaba fragments, scaled by 1/3 and 2,3, respectively. Units of AA are the experimental C.D. (absorbance units x lfls~ scaled to A 2 8 o = 1.0 for each protein.
C.D. Spectra of IgD Proteins and Fragments
395 DISCUSSION
The C.D. spectra of the intact IgD myeloma proteins studied exhibit an overall positive absorption in the 250-320 nm region in agreement with the data of Johnson et aL (1975). It is of interest to note that these features are more similar to those reported for IgE (Dorrington & Bennich, 1973) than for the other tO immunoglobulin classes (Johnson et al., 1973; Reisen et al., 1976). A feature of special interest is the positive band observed at 235 nm with protein Ai. The C.D. spectrum of an unfractionated tryptic digest of IgD protein Ha was indistinguishable from nm nm 210 230 250 250 270 290 310 3:30 that of the intact protein and thus we were unable to detect any conformational change resulting from Fig. 4. The far (A) and near (B) u.v.C.D, spectra of the cleavage in the hinge region. Similarly we show that tFc~ ( 0 - - - 0 ) , tFab~ (& A) and papain derived Fc~ the spectra of the isolated Fab~ and Fc~ fragments are additive and in sum equivalent to the intact pro(11--11) fragments of IgD protein Ai. tein (Fig. 3). It remains possible that biologically significant (Vitetta & Uhr, 1975; Konno et al., 1975) The Fab~ fragment is overall positive and clearly con- conformational changes may occur on cleavage in the tains the negative component at 300 nm seen in IgDAi hinge region that are not detectable on C.D. spectral and shows distinctive features at 292, 288 and 284 nm. analysis. Again reasonable additivity is shown for the scaled Comparison of the C.D. spectra of the Fc~ and Fc, sum of the F% and Fab6 components compared to (Dorrington & Bennich, 1973) fragments show that that of the intact protein IgDAi (Fig. 5). The deviation whilst Fc~ has positive bands at 295 nm and 288 nm here may be due to reliance on the absorption it goes negative at ca. 285 nm, and continues negative. measurements for concentration determination with However, it should be noted that the Fc, fragment scattering effects or the assumption of identical is composed of C,2, C,3 and C,4 domains and that absorption coefficients for all components leading to strong negative absorption bands in the 295-250 nm small differences. region are observed for the Fc" fragment which is In comparing the spectra of the Fab6 and Fc~ frag- essentially the isolated C,2 domain. This suggests that ments derived from IgD proteins Ha and Ai it is the C.D. spectrum of an Fc fragment composed of apparent that the spectra of the Fc6 are similar to the C,3 and C,4 domains might show positive features one another over the whole range 200-330 nm includ- similar to that observed for the Fc~ fragments. It is ing the detailed positions of the aromatic fine struc- the C,3 and C,4 domains which show the greatest ture in the region 280-300 nm. By contrast the Fab6 sequence homology with the F% fragment (Spiegelfragments are characteristically different with the berg, 1975). The studies of Ghose (1971) and Ghose Fab~ fragment derived from protein Ai exhibiting a and Jirgensons (1971a) have shown that F% has negapositive band at 235 nm, greater intensity at 270 nm tive absorption bands throughout its aromatic C.D. and a negative band at 300 nm. spectrum whilst the (Fc/t)5 fragment has positive absorption bands in the 300-280nm region which, interestingly, are shifted to lower wavelengths in the AA monomer Fc# fragment. However, the C.D. spectra of the F% fragments appear to be characteristic of 12 the IgD immunoglobulin class. The papain derived F% fragment differs from the tryptic in the presence of the hinge region of the delta chain. This hinge region has galactose and N-acetylgalactosamine present in strikingly similar proportions to that observed in lgA1 proteins (Baenzinger, 1974; Jefferis et aL, 1975b). However, whilst the polypeptide structure of the lgA1 protein contains an unusual duplicated sequence having a very high proline content (12 of 30 residues) we have found that the polypeptide of the hinge region of IgD proteins is of very different amino acid composition with a high basic amino acid content and only 3 of 30 residues to be proline (J. B. Matthews & R. Jefferis, unpubnm lished observation). No significant contribution of this 250 2to 290 hinge region structure to the C.D. absorption specFig. 5. The near u.v.C.D, spectra of IgDAi (0-----0) and trum of IgD proteins is observed since the spectra the scaled sum of the contributions of the tFca and tFab~ of the F% fragments derived from digestion with fragments (&___A) obtained from protein Ai. Units of papain or trypsin are essentially identical. AA are the experimental C.D. (absorbance units x l0 s) The C.D. spectra of the Fab6 fragments show posiscaled to A28o = 1.0 for IgDAi. tive bands in the 300-260 nm region broadly similar
396
R. JEFFERIS, J, B. MATTHEWS and P. M. BAYLEY
to those reported for Fab fragments derived from lgG, IgM and IgE (Ghose, 1971 : Ghose & Jirgensons, 1971; Dorrington & Bennich, 1973). The positive band at 235 nm observed with intact lgDx~ is present in the Fab0 fragment derived fiom il with increased intensity. The occurrence of an absorption band in this region for some immunoglobulin molecules has been reported in several studies (Cathou et al., 1968; Ghose & Jirgensons, 1971b; Johnson et al., 1974) and discussed in detail by Dorrington and Smith (1972), and Cathou and Dorrington {1975). It is thought to derive from one of the tyrosine residues present in the constant region of the kappa chain with the positive C.D. resulting from interaction between the variable and constant regions of kappa chains and is enhanced on interaction of the H and L chains. It has been stated (Cathou & Dorrington, 1975) that there is no good evidence that a comparable transition is detectable in htmbda chains. However, both IgD proteins under study in this report bear lambda chains (Jefferis, 1975a) and thus a comparable transition can be observed in some lgD proteins, at least, bearing lambda chains. Comparison of C~ and C,~ sequences (Dayhofl; 1973) reveal tyrosine residues at positions 146, 180, 193 and 199, and 145. 177 and 196, respectively. Obviously these tyrosine residues are present in very equivalent linear positions and it may be assumed that the conformation of C~ and C~. domains in intact proteins would be broadly similar, thus it would be expected that the interaction resulting in the positive C.D, band at 235nm for kappa chains might also be observed in molecules composed of lambda chains. We intend to take advantage of the possibility of isolating V.V,.C, and V.C~, fragments (Jefferis & Matthews, 1977; Jefferis, 1975a) to investigate further the interactions resulting in this C D . feature lbr lgD protein Ai. lckmm'le&tement This work was supported by a grant from the Medical Research Council.
NOTE ADDED IN PROOF
Since this paper was submitted the C.D. spectra o1 four further IgD proteins has been measured. They all gave spectra similar to that obtained with protein HA in this report; i.e. no other protein exhibited the positive absorption maxima at 235 nm.
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