California Association of Chemistry Teachers
1. H. Golly Pomona College Claremont, Calif. 91713
I I
Bence-Jones Proteins and Antibodies
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critics of our government's investment in scientific research sometime complain that funds are awarded to projects investigating trivial, untypical, or esoteric problems. It is the responsibility then of the invest,igator to explain that experience has proved that close attention to the untypical or abnormal instance often yields information of very general validity, information d i c h could be obtained in no other way. As an excellent example of this, they might cite the studies of Bence-Joncs proteins, a substance which occurs only in the urine of patients of certain rare malignant diseases. Despite their abnormal nature. these proteins may serve as a vital clue in the eventual solution of one of life's major remaining mysteries, t,he molecular basis of immunity. The clue was first discovered by a practicing physician, Dr. Watson, in London in 184.5. A patient of his, known to us now only as a rich merchant, was suffering from a disea~ewe now call multiple myeloma. It was not,iced that this patient's urine was unusually viscous, and that if certain chemical agents were added to it, voluminous amount,sof white mat,erial precipitat,ed from the solut,ion. Since this was 30 years before the professional activity years of Sherlock Holmes, this Dr. Watson had to turn t,o anot,her source to identify the strange substance. Therefore, he sent a. sample of the urine to one of the leading scient,ific investigators of England at that time, Dr. Henry Bence .Tones. With the sample, he enclosed a brief not,e describing some of the physical chemical propert,ies of the substance in the urine, and ending succinct,ly with a qnestion, "What, is it?" (I). By performing an elemental analysis Dr. ,Tones was able to demonstrate that the substance in the urine was an unusual protein. The primitive state of the prot,ein chemistry of his time precluded his finding out any more about the structure or biological function of the urinary substance, nor was he able to discover why the protein was made in such large amount,^ in the diseased state. The patient died of t,he malignancy, and for many years after that no more cases of a similar urinary substance were described. In the 1890's, however, repork were published of several patient,^ dying of a disease similar to that described by Dr. Wat,sou 40 years 56
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previously. These patients also excreted large amounts of the protein which had come to he named after Dr. Jones. In this century many instances of patients suffering from multiple myeloma have been described and investigated, and in a large fraction of these instances, the pat,ient was found to excrete Bence-Jones prot,ein, sometimes as much as 30 g of prot,ein a day. Unique Chemical Composition
Since Bence-Jones proteins can easily he obtained in large, rather pure amounts, they have been extensively investigated by many protein chemists in this century. They were found to resemble other proteins in many ways. For example, they are made up of exactly the same amino acids as are other proteins; these amino acids are linked together by the peptide bonds to form long polypeptide chains with a molecular weight of approximately 22,000. Like other globular proteins, t,lrey occur naturally folded in conformations which can he greatly disrupted by mild agents, such as heating or nonaqueous solvents. A closer look, however, reveals a very remarkable difference between Bence-Jones proteins and all other prot,eins which have been investigat,ed. I n general, we are all made up of very similar proteins. The hemogloblin in an individual's red cells, for example, is very probably exactly like that in the red cells of everyone. Although some interesting variations in structure of cert,ain proteins do occur, these variations are generally genetically inherited. Because of these inherited variations one might contain a few proteins which could be isolated from only a few of his acquaintances. On the other hand, the Rence-Jones proteins from one patient has nwer been found to be chemically identical to that made by another. Different Bence-Jones proteins vary in their amino acid composition and sequence, as well as in many other physical and chemical properties. Each Renee-Jones protein is, as far as we know, a protein which has never been synthesized before and which no one will ever make in the future. In general, all t,he molecules of the Rence-Jones protein made by a single patient are ident.ical. Berause of this individual specificity, it is common to label the different Bence-Jones proteins by the name or initials of the patient producing t,hem.
Although the proteins have served as a useful diagnostic tool and research material for protein chemists for many years, Dr. Watson's question, "What is it?" remained essentially unanswered for over 100 years. BeneeJones proteins, unlike most proteins which have enzymatic properties, do not catalyze hiochemical reactions, nor has any other structural or physiological function been assigned to them. Until 1955 no chemical relationship between BenceJones proteins and any normally occurring biochemical substance could be demonstrated. I n that year investigators applied new techniques of immuno-diffusion to the problem. Human y-globulin, a protein occurring in all normal human sera, was injected into rabbits who responded by making antibodies, proteins which would attach to specific sites on the human yglobulin and form a visible precipitate in an agar gel. When this rabbit antisera to y-globulin was allowed to react with Bence-Jones proteins a precipitate formed. This demonstrated that molecules of Bence-Jones proteins contain sites, called antigenic determinants, which are also present on human y-globulin. The fact that antisera made by injecting rabbits with Bence-Jones proteins reacted with human y-globulin confirmed this. The fact that these proteins share antigenic determinants provides very good evidence that Bence-Jones proteins are closely related to a normal human protein, yglohulin. The nature of the disease, multiple myeloma, is also consistent with the hypothesis that serum yglobulin and Bence-Jones proteins are closely related. The disease is characterized by the rapid proliferation of marrow cells which closely resemble those which synthesize y-globulin in healthy individuals, and we now know that the abnormal proteins are made in the proliferating cells. Relationship to Gamma Globulin
The chemical basis of the relationship between Rence-Jones proteins and normal y-globulin remained unclear until 1960 when G. 11.Edelman a t the Rockefeller University discovered that y-globulin molecules are not composed of a single polypeptide chain, as had been believed up to that time, but rather are made by linking together two different types of peptide chains (f). These chains are held together by covalent bonds between the sulfur atoms in the cysteine residues in the different chains as well as strong noncovalent interactions. If the disulfide links are broken by reducing or oxidizing agents, and the protein placed in a solvent which disrupts the noncovalent bonds, the component chains come apart and can be separated by a variety of techniques. Each y-globulin molecule was found to he made up of four polypeptide chains, two chains having a molecular weight of around 60,000 and designated heavy chains, and two light chains of about a third the size of heavy chains. By rombining what we know about the size and shape of these chains and the complete yglobulin molecule with the large amount of other information which investigators have learned about these substances over the years, it has even been possible to propose a tentative model for the y-globulin molecule to show how the four chains might be put together (3) This model is shown in Figure 1.
Figure 1. A proposed model for the y-globulin molecule 131. H = heavy choinr; L = light choinr. The diwlflde bonds linking the chains are shown as solid block bars. The arrow indicote the ontigen binding sites.
The fact that the chains of Bence-Jones proteins were of the same size and shape as the light chains of yglobulin suggested that the two kinds of chains were analogous. This hypothesis was confirmed by the fact that the antigenic determinants shared by 7globulin and Bence-Jones proteins were located on the light chains but not on the heavy, as well as by a great deal of other evidence which will be described later. In one property, however, the light chains of yglobulin differ markedly from Bence-Jones proteins. Unlike almost all other proteins which occur in the serum of normal individuals, yglobulin appears to be very heterogeneous, that is, made up of a population of a great many unlike molecules. Thus, if a sample of normal y-globulin is placed in an electric field, some of the molecules will consistently move faster than others, and if heated, some will denature a t a lower temperature than others. Whether these molecules differ because they are made up of different sequences of amino acids, or whether all the molecules have identical polypeptide chains which are merely folded into different shapes is very difficult to determine by studying only the normal serum protein. Light chains separated from normal y-globulin, in striking contrast to Bence-Jones proteins, also appeared to be very heterogeneous. Thus, although all the molecules of a Bence-Jones protein produced by a single individual are alike, the molecules of light chains in a single person differ a great deal in their propertics from one another. On the other hand, it is difficult or impossible to distinguish the light chains produced by one individual from those of another, whereas the Bence-Jones proteins from two different patients are never identical. One simple hypothesis to explain this state of affairs would be to presume that normal individuals possess the capability of synthesizing a very large number of different y-globulin light chains. I n multiple myeloma one of the huge number of cells making these chains might for some reason start to grow and multiply at a rate much faster than its neighboring cells. If the proliferation of this family of like cells is not stopped, and if the cells continued to synthesize only one out of the large population of normal light chains, this particular kind of polypeptide chain might be made in extraordinarily large amounts. These light chains might combine with heavy chains to form a serum protein, or they might be made even faster than heavy chains, in which case they would also occur free in the serum. Since the light chains are so small, they would pass freely through the pores in the kidney and enter the urine, where they are called Bence-Jones proteins. This hypothesis suggests that patients of multiple myeloma might have in their serum a y-globulin-like Volume
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protein containing the polypeptide chains of a BenceJones protein. I t has been known that patients of this disease often do have a large amount of such an abnormal serum protein. These abnormal proteins, called myeloma globulins, have the same size and chain structure as y-globulin, but differ in that they are far more homogeneous. Dr. Edelman and I decided to isolate the light rhains from a myeloma globulin and compare them to pzlypeptide chains of the Hence-.Jones protein made by the same patient. We found that the peptide chains from the two sources contained the same amino acids in the same proportions, that the chains possessed the same antigenic determinants, and that they both migrated at the same velocity in an electric field (4). Further work demonstrated that the two proteins would break down to identical small fragments when heated with the enzyme trypsin which breaks peptide chains following the basic amino acids. This teohnique, called fingerprinting, is a powerful tool for discovering differences among proteins. All our results indicate that Bence-,Jones proteins are indeed identical to the light chains of the myeloma protein made by the same patient. This work when added to that of many other workers yields a convincing and consistent picture which relates the physical chemical properties of Bence-Jones proteins and their appearance in multiple myeloma with the other clinical manifestations of the disease. Thus in large measure we may give a satisfying answer to Dr. Watson's question, "What is it?" which had remained such a puzzle for over 100 years. Even more important, however, these advances yield new information about the structure and synthesis of yglobulin and as a result tell us a great deal more about one of nature's best kept secrets, the nature of the immune response. Immunity and Antibodies
To appreciate the extent of this mystery, consider again those rabbits which were injected with a BenceJones protein and which responded by making a new serum protein, an antibody, capable of specifically attaching to antigenic sites of the protein and precipitating with it. Almost any protein which does not normally occur in the rabbit would call forth an analogous response when injected into the rabbit, that is, antibodies would appear in the rabbit's bloodstream which had not previously been detectable and which would attach specifically to the injected foreign protein, known as the antigen. Antibodies which are elicited by the injection of any one antigen will not, in general, hind to any substance which is not very similar in couformation to that antigen. The number of different foreign substances which the body is capable of responding to by making specific antibody is a matter of much controversy, but it is surely very large. Most immunochemists would agree that antibodies can distinguish among a t least 10,000 different antigens; many would say that a single organism must be capable of making antibodies of over a million different ~pecificit~ies. A11 antibodies belong to a class of serum proteins called immunoglohulins, most of them belong to the major sub-group of the immunoglobulins, y-globulin. 58
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A small proportion of all antibody molecules, around lo%, differ from y-globulin molecules in a number of properties; molecular weight, electrostatic charge, etc., but even these contain light chains like those in Bence-Jones protein. Indeed, light chains are the only structural unit common to all immnnoglobulins. The biological funct,ion of the antibody response is well known. Viruses, toxins, and other potentially harmful substances can act as antigens when they invade an organism and the antibodies made in response can often neutralize the harmful effect of the foreign material by specifically combining wit,h it. The efficiency of this defense mechanism, found in almost all vertebrates, is testified by the great success of immunization programs against sn~allpox, polio, and many other diseases. By injecting the innocuous form of the microorganisms which cause these diseases we are able to stimulate the production of antibodies which serve to neutralize the virulent microbe which may later get into our system. Although the use of immunization in preventive medicine has been practiced for over 1.50 years, the molecular basis of the phenomena remains a mystery. Consider what the system involves. .411 organism must be able to distinguish a foreign iuvader from the thousands of other substances normally preseut in the body. Then the body must be able to synthes'ze in large amounts a protein which is capable of binding to the invaders with which the body has never before been in contact. I t might be thought that the enormous increase in our knowledge about the structure arid synthesis of proteins gained in the last decade might have helped to clarify the basis of the formatioo of specific antibodies, but in many respects this increased knowledge raises more new questions than it answers. Certain principles of protein chemistry have been shown recently to have such general validity, however, that it is widely assumed that any mechauism of antibody formation must conform to these principles. For example, recent advances in protein chemistry support t,he hypothesis that the manner in v4iich a protein folds in space is determined solely by its primary structure, that is, by the number of amino acid residues in the protein and the sequence in which they are arranged. I n accordance with this it has been shown that the conformation of antibody molecules can be completely unfolded by placing the protein in an unusual, disrupting solvent, but if the protein is returned slowly to a water solution, the native conformation and ability to bind antigen are spontaneously regained (5, 6'). This indicates that the amino acid sequence itself contains enough information to form a specific antibody, and that the autihody molecnle does not "mold" itself upon the antigen as had previously been suggested. Another hypothesis now generally accepted concerning protein structure states that the sequence of amino acids in all the body proteins is coded for in the sequeuce of nucleotides that makes up the DSA stored in the chromosomes of the cells. This hypothesis suggests that the information to synthesize all the different type antibody molecules a given organism can make is stored in the DNA component of that organism. A major problem the0 to solve would be to discover how a
given antigen can result in the body's selecting the correct stretch of DXA which codes for the correct antibody. The elucidation of the nature of Bence-Jones proteins gives added support to these two major hypotheses of antibody structure and formation. Physical chemical studies of a great many different types of Bcnce-Jones proteins prove that light chains can vary in their amino acid sequence a great deal among individuals and, therefore, they suggest that a single individual may produce a great variety of light chains. Studies have also shown that the conformation of different pure light chains differ from one another, and that this conformation is determined solely by the primary structure. In addition, the fact that the same Rence-Jones protein is made by all the malignant cells in a multiple myeloma patient suggests that the information for synthesizing that protein must reside in the genetic material of those cells. As mentioned above, antibody molecules occur in the y-globulin fraction of the serum proteins. All antibody molecules c,ontain light chains indistinguishable by physical chemical criteria from those of normal yglobulin and analogous to those of Bence-Jones proteins. We know that antibody molecules of different specificities vary one from another becausc thcy bind to different antigens, but the chemical basis of these differences is unclear. I t has proved impossible to obtain a homogeneous, pure sample of any antibody, nor is it easy to see how it will ever be possible to obtain such a sample, so the determination of the primary sequence of an antihody molecule is at the present time impossible. If we accept the premise that the light chains of antibodies of different specificities diffcr in the same way t,hat different Bence-Jones proteins differ, however, by studying Bence-.Jones proteins we may discover those features of antibody molecules which permit them to hind specifically to an antigen. Myeloma Globulin a Clue to Antibody Biosynthesis?
The importance of the studies of Bence-Jones proteins at this time is based on these considerations. We are unable to obtain large amounts of pure antibody for performing chemical studies, so nature provides us a substitute in large, homogeneous amounts. Bence.Jones proteins are not antibodies, but they appear to bc very closely analogous to natural sub-units of antibodies, and they do lend themselves to the complete elucidation of their structure. I t might he argued that although antibody molecules contain light chains, these chains do not contribute to the immunologic activity of the antibody, and that the activity is determined solely by the structure of the heavy chains. Many workers in several laboratories have been studying the problem in many different ways, and the general conclusion of this work has been that both chain types are present at the antibody comhing site, as shown in Figure 1, and that both contribute to the specificity of that site (7-9). In agreement with this conclusion it has been shown that both the light and heavy chains isolated from an antibody preparation directed to one antigen appear to be chemically different from the light and heavy chains of an antibody molecule of a different specificity. Convinced that t,he knowledge of the primary se-
quence of the Bence-Jones proteins would contribute a great deal to our understanding of the structure and biosynthesis of antibodies, a number of groups have undertaken the arduous task of finding out in what order the 214 amino acid residues which make up these polypeptide chains are linked toegther (10-15). The unexpectedness of their results have already more than justified the great effort they have expended in obtaining this information. So far none of this work is complete, that is, the complete amino acid sequence of any one Rence-Jones protein has not yet been determined. Since no two of these proteins are identical and since different workers are investigating proteins obtained from different sources, it could not be expected that they will come out with identical sequences. A number of very striking similarities and differences between the sequences of the different proteins studied by the separate groups have emerged already, however, which indicate that these proteins are interrelated in a fashion which makes them altogether distinct from other proteins. As indicated in the diagram in Figure 2 it was found VARIABLE
CONSTANT
Figure 2. Schematic diagram of the primary structure of the light chains of Bence-Jones proteins. The solid line represenh the polypeptide backbone which is made up of appraiimotely 214 amino acid residues. The doned lines represent the internal diwlflde bonds. The free rulfhydryl group odimcent to the corboxy terminus i, dirulflde bonded to heavy chains in y-globulin. The primory sequence of the CONSTANT portion of the polypeptide chain is nearly identicol in ail Bence-Jones proteins, the VARIABLE region is quite different in each protein.
that approximately half of the amino acid sequence tentatively proposed for one Bence-Jones protein appears to be identical to the homologous region of other Bence-Jones protein molecules. The other half of t,he sequence, those 107 amino acids nearest the free amino end of the polypeptide chains, show a great deal of individual variation from one Bence-Jones protein to the next. In some sense these results fit in well with our concepts of antibody structure. Experiments have clearly shown that light chains from many different sources can bind to different heavy chains to form one-to-one complexes like those of naturally occurring 7-globulin. This would suggest that there might be some surface feature on all light chains which permits them to bind to a site common to all heavy chains. A common binding site on all light chains would suggest that at least some portion of their amino acid sequence could be similar. That they would have some portion containing unlike amino acid sequence is implied by the hypothesis that the structure of light chains influence the specificity of antibodies. Although the hypothesis that light chains contain regions with similar amino acid sequences as well as regions with differing sequences fits very well into our model of the antibody molecule, other considerations make the results of the sequence determination very intriguing indeed. If antibodies are synthesized like other proteins, and some evidence suggests they are, then the sequence of Volume 44, Number 7, January 1967
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amino acids in the light chains must be coded in a sequence of bases in the DNA of the cell nucleus, as well as in the messenger RNA molecules which transports the sequence information from the chromosomes to the protein-manufacturing sites in the cell. It is difficult to see how it could be possible for one half of a strand of RNA to vary the base sequence on one end while keeping the sequence on the other end constant without postulating some very unorthodox genetic mechanisms. The nature of the variation in the amino-terminal half of the molecule is being closely examined for clues as to how the variability is generated. Although the outcome of these studies is not yet clear, it is already obvious that many of the hypotheses proposed to explain the sources of variations in the structure of antibodies do not agree with this sequence data. It is probable that new theories will have to be proposed to account for the novel sequence variation found in these proteins. Although the work on the structure of Bence-Jones protein might be extremely enlightening, it surely will not reveal all we want to know about the structure of antibodies. Heavy chains contribute more on the basis of mass than do light chains to the antihody molecule, and the structure of heavy chains clearly have a major influence on antihody specificity (57). In several ways heavy chains are analogous to light chains. For example, heavy chains isolated from 7globulin molecules appear to have a common amino acid sequence, and also regions of diversity. The heavy chains normally present in our bodies are very heterogeneous, hut in multiple myeloma they can be produced in large, homogeneous amounts, as a part of myeloma globulins. Preliminary studies on the amino
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acid sequences of these homogeneous heavy chains preparations have already begun. It is a safe assumption that what we learn about myeloma globulin heavy chains will teach us facts about the structure of antibodies we could discover in no other way. To those of us who try to learn more about the workings of nature she often seems almost wilfully secretive. At other times, however, she seems to delight in doing experiments for us. As far as we can tell, Bence-Jones proteins perform no other useful function than helping solve the mystery of our mechanisms of immunity. By exploiting this clue properly, man may sometime learn a great deal he can use to improve his health. Literature Cited (1) JONES,H. E., Phil. Trans. Roy. Sac. London, 138, 55 (1848). (2) EDELMAN, G. M., AND POULIK,M. D., J. Ezp. Med., 113, 861 (1961). (3) EDELMAN, G. M., AND GALLY, J. A,, Proe. Natl. Acad. Sci. U.S., 51, 846 (1964). (4) EDELMAN, G. M., and G A I ~J., A., J. E z p . Med., 116, 207 119fi21. ~ . ..(5) H ~ E R , E . P , r o ~Natl. . Acad. Sci., U.S., 5 2 , 1099 (1964). (6) WHITNEY, P. L., AND TANFORD, C.,Pmc. Natl. Acad. Sci. U S . , 53, 524 (1965). G. IM., O I ~ N SD. , E., GALLY, J. A,, AND ZINDER, (7) EDELMAN, N. D., Proe. 1Vatl. A d . Sei. U S . , 50, 753 (1963). (8) METZGER, H., WOFSY,L., AND SINGER,S. J., P ~ c .ha. . Acad. Sci. U.S.. 51. 612 (1964).
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ROHOLT, O., ONOUE,K., AND PRESSMAN, P., PTOC.~ V a f l . A d . Sci. U.S., 51, 173 (1964). HILSCHMANN, W., AND CRAIG,L., P ~ c Natl. . Acad. Sci. U.S., 53, 1403 (1965). T I ~ N K., I , WHITNEY, E., JR., AVAGARDO, L., AND PUTNAM, F. W.. Seiace, 149. 1090 (1965). (12) HOOD,L. E., GUY, W. R., AND DREYER, W. J., Proc. .Vall. Acad. Sci. U S . , 55, 826 (1966). (13) hfrr.sTEIN, C.,Nature, 209, 370 (1966).
