Subunit structure of human γM-globulins

lmmunochemistry. Pergamon Press 1970. Vol.7, pp. 651-660. Printedin Great Britain

SUBUNIT STRUCTURE OF HUMAN TM-GLOBULINS* K. J. DORRINGTON and C. MIHAESCO M. R. C. Molecular Pharmacology Unit, Medical School, University of Cambridge, United Kingdom and Laboratory of Immunochemistry, Research Institute on Blood Diseases of the University of Paris, Hospital Saint Louis, Paris 10, France

(Received5January 1970) Abstract-The molecular weights of an intact Waldenstrom TM, its heavy (/z)-chain and the various fragments produced by papain and pepsin have been determined by sedimentation equilibrium. The homogeneity of the purified proteins has been confirmed by studies of the dependence of the weight-average molecular weights on initial protein concentration and rotor speed. The results confirm earlier evidence locating the principal antigenic determinants of yM in discrete regions of the molecule. The/z-chain has been delineated into three, non-overlapping regions, of different molecular size, each showing characteristic antigenic properties. The total mass of 3,M can be accounted for by two proteolytic fragments (pepsin F(ab")z and papain Fc). The results provide further evidence for a model of ~/M consisting of five subunits each made up of two heavy and two light chains. INTRODUCTION The recent progress in understanding the molecular structure of yMglobulins is mainly due to physicochemical and immunological studies of their subunits obtained by 'longitudinal' cleavage (inter chain disulphide and noncovalent bond cleavage) and by 'transverse' cleavage (limited proteolysis) of the native molecule. These approaches have led to a schematic representation of the ~/M molecule consisting of five monomeric subunits, linked by disulphide bonds, each consisting of two heavy (/z) polypeptide chains and two light (L) chains [1, 2]. Previous comparative studies of the papain and peptic split products of several Waldenstr6m yM-globulins permitted delineation of the native molecule into three principal regions [3-5]. The first region corresponds to the papain Fab/Z and pepsin Fab'/Z fragments and contains the L-chains disulphide bonded to the N-terminal portion of the/z-chains (Fd/Z). The second area, destroyed by papain but not by pepsin under mild conditions, is present in the peptic F(ab")2/z fragment and corresponds to the 'hinge region' of the/z-chain (by analogy with y-chains). The third area is represented by a large papain Fc/z fragment consisting of the disulphide-linked C-terminal regions of the/z-chains. This paper reports the molecular weights of the subunits and fragments of yM globulin. These studies provide a quantitative check on the contribution of these subunits and fragments to the overall structure of 3,M previously delineated by immunochemical methods. Moreover, as the papain Fc/z and peptic fragments are obtained in lower than theoretical yields, a knowledge of their *Supported in part by Grant No. C-R66237 of the French National Institute for Medical Research (I.N.S.E.R.M.). 651

652

K. J. DORRINGTON and C. MIHAESCO

molecular size is essential to any proposed molecular model for yM based on these fragments. MATERIALS AND METHODS

Preparation of yM, subunits andfragments Immunochemically pure yM(D) was isolated from the plasma of a patient with Waldenstr6m's macroglobulinemia as described previously[4]. For the molecular weight studies the purified yM was further chromatographed on a Sepharose 6B column (2"5 x 88 cm) in 0-1 M TRIS/HC1, 0.5 M NaCI, 2mM EDTA pH 8.0 to eliminate higher molecular weight yM aggregates. Polypeptide chains yM(D) were prepared according to Fleischman et al. [6] by reduction in 0.1 M 2-mercapto-ethanol at pH 8.0 and alkylation with 10 per cent molar excess of iodoacetamide. Light and /.~-chains were separated by gelfiltration on Sephadex G-100 equilibrated with 1.0 M acetic acid at 4°C. Only the mid-third of the first peak eluted was selected for the/z-chain preparation. A portion of these mildly reduced/~-chains were completely reduced with 0" 1 M dithrothreitol (DTT) in 6.0M guanidine HC1 at pH 8.6 for 4 hr followed by aklylation with 10 per cent excess iodoacetamide. The /z-chains were further purified by gel filtration on Sephadex G-200 in 6.0M guanidine HC1, 1.0M acetic acid. The papain fragments Fab ~ and Fc /~ were prepared and purified as described previously [4]. To obtain Fc/~ monomeric subunits native Fc/z fragment was reduced with 5mM D T T for 30 min at pH 8.6 and 23 °, followed by aklylation with 10 per cent excess of rodoacetamide for 15 rain then dialysed overnight against 1.0 M acetic acid at 4 ° before freeze-drying. Peptic Fab'/~ and F(ab")2/z-fragments were obtained by digestion of yM(D) for 2 hr as described elsewhere [5]. Under these conditions the yield of F(ab")2 was 16-20 per cent of the original protein. For molecular weight studies the papain Fab' and peptic Fab'/z and F(ab")/z fragments were rechromatographed on Sephadex G-200 (column 2.0 x 100 cm) in molar acetic acid and the mid-portion of the eluted peaks selected.

Molecular weight determinations Sedimentation equilibrium experiments were performed on a Beckman Model E ultracentrifuge equipped with electronic speed control, interference optics and a RTIC temperature control unit. Cells with either 12mm double sector aluminium filled epoxy resin or KeI-F six-channel centrepieces and sapphire windows were employed. The six-channel centrepiece[7] was used routinely for concentration dependance studies. All runs below 17,000 rev/min were made with an AN-J rotor, in order to minimize precession. At higher speeds, an AN-D rotor was used, and blurring of the fringe pattern which occasionally developed at high speeds (above 30,000 rev/min) was compensated for by a polarizing filter placed directly over the light source in addition to the Kodak 77-A filter. Measurements of fringe displacements against radial distance were made with a Nikon comparator. Fringe displacements in the double sector cells were water blank corrected for window distortion as described by Yphantis [7]. The system was j u d g e d to be at equilibrium when the fringe displacement at

yM Structure

653

the solution-fluorocarbon junction remained constant for a period of 5-10 hr. Column height and rotor speed were adjusted to enable the use of the meniscus depletion method developed by Yphantis[7]. The quantity Mw app (1-~p) was calculated from the slopes of linear plots of the fringe displacement (microns) against radial distance (cm) squared, R ~, according to the relationship. d logf2RT

Mwapp ( 1 -- ~p) =

dR 2 t02

Apparent molecular weights were calculated from this quantity using the appropriate density and values for the partial specific Volume, ~. Proteins were freeze-dried and appropriate amounts dissolved in 6.0M gnanidine hydrochloride and extensively dialysed against the solvent. Final protein concentrations for yM and fragments were determined by optical 1% density measurements at 280 m/z using an extinction coefficient (Elcm) of 13"5 determined from dry-weight analyses on the native yM. For some proteins which had not been extensively reduced and alkylated, iodoacetamide (0.02 M) was added to the guanidine solution to minimize disulphide interchange and subsequent aggregation. Failure to do this invariably resulted in curvature in the fringe displacement vs. radial distance plots. Solvent densities were determined either pycnometrically or from refractive index measurements. The values of V used to calculate M~app were as follows: yM, 0.712:/z-chain 0.710; F c 0.705; Fab' and Fab', 0.715; F(ab"), 0.713. These values were obtained from amino acid and carbohydrate analyses and corrected for preferential guanidine binding by subtraction of 0.01 ml.g -1 (for discussion see Montgomery et al. [8]). RESULTS Equilibrium sedimentation of intact yM(D) in 6.0M guanidine 0.02M iodoacetamide yielded linear plots of logfvs. R 2 giving a molecular weight near 890,000 (Fig. 1). This linear plot together with the lack of significant dependence of Mwapp on rotor speed or initial protein concentration (Table 1) provides strong evidence that the ~/M was homogeneous. In all cases where valves of Mwapp were independant of speed and protein concentration the M~ app values were averaged to give . ~ 0 (the weight-average molecular weight at zero protein concentration). The omission of iodoacetamide from guanidine solutions of yM and its fragments almost invariably led to significant curvature in the l o g f vs. R ~ plots and higher values for Mwapp. This phenomenon is thought to be due to the exposure of buried-SH groups on unfolding in guanidine solutions leading to inter- and intra-molecular disulphide interchange, the former leading to aggregation. The extensively reduced and alkylated p.-chain had a molecular weight near 65,000. Again the insensitivity of the apparent molecular weight to changes in rotor speed and protein concentration suggested a high degree of mass homogeneity (Table 1). The log f vs. R 2 equilibrium plots for papain Fab/z and pepsin Fabl/z are shown in Fig. 2. The linearity of these plots together with data obtained at different rotor speeds and initial protein concentrations (Table 1) suggest that

654

K . J . D O R R I N G T O N and C. M I H A E S C O

M(Olin

3"2

/ /I/ . , ,

guonidin¢

--c ~ 2"8

m~~///// plus iodoaccta

E

2"/. "Oo ~ 2"0 . //minus

/,9.5

I

iodoacctamid=

I

50.0 50-5 51-0 Radial DistanceSquared(cmz)

Fig. 1. T h e equilibrium distributions of intact TM(D) in 5'98 M guanidine HCI with and without 0"02 M iodoacetamide after 40 h r at 8,000 rev/min. T h e logarithm o f the fringe displacement in microns is shown as a function of the square o f the radial distance in centimeters. T h e vertical line, m a r k e d rb, represents the position of the solution-fluorocarbon oil interface: Table 1. T h e d e p e n d e n c e o f the a p p a r e n t weight-average molecular weights of TM(D), /~-chain and various proteolytic fragments on rotor speed and initial protein concentration Mwapp × 10-3 at protein concentrations Protein 7M a /z-chain b Fab (papain) a Fab' (pepsin) a F(ab")2(pepsin)~ Fe polymer a Fc m o n o m e r c

Rotor speed (rev/min)

0.25

0.5

0.75 mg.m1-1

M o o ± S.D.

(× 10-a)

8,000 10,000 24,000 28,000 26,000 28,000 26,000 28,000 16,000 20,000 11,000 13,000 34,000 40,000

867-0 912"0 65' 1 64.5 45"7 44"2 46"7 47"3 116.0 119.9 308"9 312"4 32-7 31"6

900.0 874"0 62"4 65"9 43"6 46.3 50" 1 48.5 129.7 116.8 325"0 311.7 29'3 32.1

877"0 916"0 68"0 65"3 46"9 43-9 48.8 47-8 112.8 121.6 309"7 318.3 31"7 32'0

891 "0 ± 20"6

aSolvent 5.98 M GuHCI/0.02 M iodoacetamide p H 8.0. bSolvent 6.3 M GuHCI p H 8"0. eSolvent 5.98 M GuHC1/0-1 M 2-mercaptoethanol p H 8"0.

65"2 ± 1"8 45.1 ± 1-6 48"2--_ 1"2 119.4±5.5 314-3 ± 6"2 31-5___ 1.1

655

TM Structure -

w

-

,

-

iI

/

Pepsin F (ab") 2 p

"~c uo "~

t 6,000 rpm

O.Srng.ml7' /

/

/

2./)

-~-02.0/

/ I

t, 8"0

20,000 rpm

//

.

02.Sm mgt~. ,

I

,

t,9-O 50"0 Radial Distance Squared (cm 2}

I

51-0

Fig. 2. The equilibrium distributions after 35 hr of F(ab'%/z at rotor speeds and two initial protein concentrations in 5.98M guanidine HC1/0"02M iodoacetamide. Other details as in Fig. 1. both fragments are homogeneous. Slight differences in Mw° are apparent: 45,000 for Fab/~ and 48,000 for Fabl/z. This difference, however, is of the same order as the error in the determinations and may be due to this error or to the choice of the same ~ value for both fragments. The use of a similar ~ value may not be justified in view of the small differences in carbohydrate content between the two fragments. The pepsin-produced F(ab")2/z fragment had a molecular weight near 119,000 and was homogeneous by the criteria outlined above for the other fragments (Fig. 3; Table 1). Sedimentation equilibrium studies were performed on the papain Fe/z fragment before and after reduction. The polymeric Fdx had a molecular weight of 315,000 in 6.0 M guanidine solution (Table 1). In dilute salt solution the protein showed a tendency to polymerize which was not apparent in guanidine solutions (Fig. 4). Equilibrium sedimentation of Fdx in 6-0M guanidine and 0.1M 2mercaptoethanol yielded a molecular weight near 31,000. Mild reduction and aklylation of Fdz prior to equilibration in guanidine solution yielded a similar molecular weight. In both instances reduced Fdx gave linear plots of logfvs. R 2 suggesting quantitative conversion of polymeric Fc to its monomeric units (Fig. 4). DISCUSSION The topographical relationship between the various fragments and polypeptide chains examined in the present study and those produced by trypsin [9] and chymotrypsin C [10] is shown in Fig. 5. The molecular weight (891,000) of yM(D) is close to previous values for other Waldenstr6m macroglobulins[1, 10]. However other workers have provided

656

K . J . DORRINGTON and C. MIHAESCO 3'2

Fab'.--,,~ .A

2.8

o

u E "-o 2.4

o /

26,000 rpm

2.0 !

i

Fob'

36-0

37-0

36-0

Fob

50.0

51"0

52.0

Radial Distance Squared (cm 2 )

Fig. 3. A comparison of the equilibrium distributions of papain Fab/z and pepsin Fab' after 36 hr at 26,000 rev/min. The initial protein concentration was the same for both fragments. Other details as in Fig. 1. F c - p o l y m t r , 0"25rag.nll"I in guanidin¢/iodoclceta m i d ¢ / ~ [

Fc -monomer,0.25rag.ml-I in 9uonidin¢/mcrcaptocthanol //~/rb

3"2

2"8

Z , 2"0 I.

i

49"0

50.0

Radial

I

I

51 0 /.9.0 50.0 Distance Squared (cm 2)

I

51.0

Fig. 4. The equilibrium distributions of Fe~ in 5"98M guanidine HCI with and without 0-1 M 2-mercaptoethanol after 35-40 hr at each indicated rotor speed. Other details as in Fig. 1. evidence for a wider r a n g e o f values (620,000-1,200,00) for intact TM and the reductive subunit yMs[11, 12]. We c a n n o t provide an adequate explanation for these discrepancies at this j u n c t u r e . While these differences might be d u e to the presence o f the known subclasses of/z-chain [13, 14] or the subgroups o f TM with different c a r b o h y d r a t e content[15], they seem to be outside the anticipated variation for which such differences would account. O u r value o f 65,200 for chain c o m p a r e s well with previously r e p o r t e d values for h u m a n [ 8 , 16] and rabbit[17] /z-chain and is substantially h i g h e r than the

yM Structure

657

PAPa,IN

,

-.Iq~

~

/

I

/

.-"~---.~v--_"

*,

I

s-, (3~s.ooo)

!

--.

~ ' ' /

.',,-~-~"~ ,

..,

~

"JO

~

~1~7-"

Fig. 5. The proposed model for yM based on the molecular weight data for the papain, peptic, tryptic[9] and chymo-tryptic [10] fragments and reductive subunits. The molecular dimensions are taken from the electron microscope data. For further details see "Discussion". reported values for y-chain [18, 19]. This difference can only be partially accounted for by the higher carbohydrate content of/z-chain compared with y-chain. The carbohydrate of yM(D)/z-chain accounts for 9000 g.mole -1 leaving a polypeptide portion of 56,200. This compares with near 48,600 for the y-chain polypeptide [20]. Although the molecular size of the kappa chains of yM(D) were not measured in this study, previous data obtained by one of us [8] indicated a value of 23,500. This value has been used for calculations associated with the present study as the light chain represents a common structural element of all immunoglobulin classes. The Fab/z and Fab'/z-fragments split from the N-terminal region of yM by papain and pepsin respectively are chemically similar but not identical. Although their antigenic identity was supported by immunodiffusion with several antisera and by cross-absorption experiments, studies with antisera raised against each

658

K.J. DORRINGTON and C. MIHAESCO

type of fragment were not performed. Their chemical non-identity is indicated by the greater electrophoretic mobility of Fabix and its lower carbohydrate content compared with Fab'ix. We have now shown that the Fabix may have a slightly greater molecular size (48,200) than Fab'ix (45,100) which could account for the noted chemical differences. The molecular weight of near 120,000 for F(ab")2 is consistent with the immunochemical properties of this fragment indicating the presence of two L chains, each disulphide linked to a segment of Ix-chain (Fd"Ix piece). The two Fd'Ix pieces are linked by a single disulphide bridge. Trypsin digestion o f y M yield a fragment of similar molecular size (114,000) and immunochemical properties [9] while chymotrypsin C produces a larger fragment (135,000)[10]. It can be easily calculated that each Fd"Ix piece of 36,200 mol. wt. is larger than Fd Ix and Fd'Ix by approximately 14,600 and 11,500 g-mole -1 respectively. These additional Ix-chain segments, very sensitive to proteolytic attack, are reminiscent of the 'hinge' region of yG-globulins [21] but are more extended in yM. The antigenic study of this region suggested only a low level of conformational structure present at this level. Its primary structure may well indicate a constant region of the Ix-chain, as several Fabix and Fcix absorbed antisera against several different yM-globulins reacted equally well with this region. The disulphide bridge linking the two Fd"IX pieces is probably located in this peptide. The fact that F(ab")2Ix can be dissociated in Fab"Ix monomers on mild reduction in neutral aqueous solution indicates that the non-covalent forces between the Ix-chains are very weak at this level. Our value for the molecular weight of the large papain Fc-fragment (314,000) is close to that obtained by Onoue et al. [22] for a similar polymeric fragment from yM. The Fc lacked any antigenic determinants present on the F(ab")2Ix fragment and is made up of several disulphide-linked Ix-chain segments. The C-Terminal sequence of these monomeric Fc units (i.e. cys-tyr) is identical to that of the corresponding Ix-chains [23,. On reduction the molecular weight of the Fc-fragment dropped to 31,500 in 6 M guanidine indicating that the polymeric Fc is made up of ten monomer units. Each Fc monomer contains two blocked half-crystine residues following mild reduction and alkylation; each derived from different interchain disulphide bridges. Following selective cleavage of interchain disulphides each Ix-chain contains four half-cystine residues [23]. The first of these (from the N-terminus) is involved in the L-IX disulphide bridge and is found in the Fd'/Fd'IX region. The second participates in a/x-Ix bond in the putative 'hinge' region and is located in the Fd'IX region. The other two halfcystines are located in the Fc region, one being the penultimate from the Cterminus. This half-cysteine is involved in a intra-yMs, inter Ix-chain disulphide bond and the second, more N-terminal residue forms a disulphide bond linking the T M subunits ([24]; Fig. 5). Our molecular weight values for the TM fragments and chains allow us to calculate a molecular weight for yMs of 177,400. This value is consistent with that obtained from the intact yM assuming five subunits i.e. 891,000/5 = 178,200, and in keeping with the data obtained by Miller and Metzger [1] using sedimentation/ diffusion analyses. The molecular weights of the Fabix fragment and intact 7M can be used to

yM Structure

659

calculate the theoretical yield of Fab/z following papain digestion. T h e experimental yield of this stable f r a g m e n t (47-50 per cent) is close to the theoretical yield (54 per cent) a n d indicates the presence of ten Fab/z fragments in the intact molecule. T h e experimental yields of papain Fc and pepsin F(ab")2-fragments are lower than theoretically anticipated therefore an estimation of their contribution to the molecular weight of yMs is relevant. Assuming that two monomeric Fc/Z segments and one F(ab")dz f r a g m e n t contribute to yMs, the sum of their molecular weights is 182,000 (i.e. 119,000 + (2 × 31,500)). This value is essentially the same as that calculated f r o m the polypeptide chains (178,000) since errors are being multiplied in this type of calculation. We can conclude, therefore, that these two types of f r a g m e n t alone can account for the whole yM molecule without introducing additional moeities. T h e present data strongly support the 10 L-10 /z-chain structure of yM. F u r t h e r support for this model has been obtained f r o m electron micrographs of yM which indicate a five-armed star-shaped structure for this molecule [25]. T h e central area of the molecule representing the Fc region displays a ring structure [26]. T h e dimensions o f the yM molecule a n d its fragments f r o m electron micrographs are in good a g r e e m e n t with the present molecular weight values, in view of the f u n d a m e n t a l differences between the two approaches. We can find no evidence f r o m o u r data to support the alternative model which proposes a 15 L10/z-chain structure for yM [16, 27].

I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

REFERENCES Miller F. and Metzger M.,J. biol. Chem. 240, 3325 (1965). Miller F. and Metzger H.,J. biol. Chem. 240, 4740 (1965). Mihaesco C. and Seligmann M., C. r. hebd. S~anc. Acad. Sci., Paris 262, 2661 (1966). Mihaesco C. and Seligmann M.,J. exp. Med. 127, 431 (1968). Mihaesco C. and Seligmann M., Immunochemistry 5,457 (1968). Fleishman J. B., Pain R. H. and Porter R. R., Archs Biochem. Biophys. Suppl. 1, 174 (1962). Yphantis D. A., Biochemistry 3, 297 (1964). Montgomery P. C., Dorrington K.J. and RockeyJ. H., Biochemistry 8, 1247 (1969). Miller F. and Metzger H.,J. biol. Chem. 241, 1732 (1966). Chen P. J., Reichlin M. and Tomasi T. P., Jr., Biochemistry 8, 2246 (1969). Filitti-Wurmser S. and Hartman L., Revuefr. etud. clin. biol. 13,967 (1968). Filitti-Wurmser S., Tempete-Gaillourdet M. and Hartmann L., C.r. hebd. S(anc. Acad. Sci., Paris 269, 513 (1969). Franklin E. C. and Frangione B.,J. Immun. 99, 810 (1967). Franklin E. C. and Frangione B., Biochemistry 8, 4203 (1969). DavieJ. M. and Osterland C.,J. exp. Med. 128, 699 (1968). Suzuki T. and Deutsch M. F.,J. biol. Chem. 242, 2725 (1967). Lamm E. M. and Small P. A.,Jr., Biochemistry 5,267 (1966). Pain R. M., Biochem. biophys. Acta 94, 183 (1965). Piggot P. J. and Press E. M., Biochem.J. 104, 606 (1967). Edelman G. M., Cunningham B. A., Gall W. E., Gottlieb P. D., Rutishauser V. and Waxdal M.J., Proc. hath. Acad. Sci. U.S.A. 63, 78 (1969). Smyth D. S. and Utsumi S., Nature, Lond. 216, 332 (1967). Onoue K., Kishimoto T. and Yamamura Y.,J. Immun. 100, 238 (1968). Mihaesco E. and Mihaesco C., Biochem. biophys. Res. Commun. 33, 869 (1968).

660 24. 25. 26. 27.

K. J. DORRINGTON and C. MIHAESCO Mihaesco C., Frangione B. and Franklin E. C., Fedn Proc. In press (1970). Chesebro B., Bloth B. and Svehag S. E.,J. exp. Med. 127, 399 (1968). Svehag S. E., Bloth B. and Seligmann M.,J. exp. Med. 130,691 (1969). Suzuki T., Immunochemistry 6,587 (1969).