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Introduction |
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Nisonoff grew up within a close-knit Jewish family in South River, New Jersey. Of college age during the depression years, he recalled having to hitchhike to classes at Rutgers where he received a B.S. in Chemistry in 1942 at the age of 19. For about a year he did war-related work at U.S. Rubber in Detroit, MI, and then enlisted in the U.S. Navy. Following discharge, he took advantage of the G.I. Bill to enroll in graduate school at Johns Hopkins University and received the Ph.D. in 1952; his thesis was an analysis of the kinetics of enzymatic transamination. There followed two additional years of research at U.S. Rubber in Connecticut. In 1954 he joined the laboratory of David Pressman, who was then at the Roswell Park Memorial Institute in Buffalo, NY, and began the work in immunochemistry that set the direction for much of his research for the remainder of his career.
In the Pressman laboratory, Nisonoff explored the interactions of antibodies with haptenic determinants, typically using the quantitative methods that remained a hallmark of his work. In an important paper from this period he demonstrated that the two combining sites on a single antibody molecule have the same specificity. This result, which confirmed earlier experiments by Karl Landsteiner, Felix Haurowitz, and Herman Eisen, was important in that it rendered it unlikely that a simple template or folding molecule, as proposed by Linus Pauling, could be responsible for generating antibody specificity. In subsequent work, he applied anti-hapten antibodies skillfully and productively to a number of problems in immunology.
Toward the end of his stay at Roswell Park, Nisonoff initiated experiments on the enzymatic cleavage of rabbit antibodies, which contributed importantly to the growing understanding of their structure. Rodney Porter had shown that two active univalent fragments, now known as Fab, could be produced from each antibody molecule by digestion with papain. Since papain is always used in the presence of a mercaptan, Nisonoff originally proposed that two steps, proteolysis and disulfide cleavage, were needed to generate the active univalent fragments. Accordingly, he repeated Porter’s experiment with a different enzyme, pepsin, which does not require activation by a mercaptan. As Nisonoff has pointed out (2), the initial premise was incorrect, but the experiment led to an even more interesting result. During cleavage by papain, a mercaptan is not required to produce active univalent fragments; however, reduction of interchain disulfide bonds is required to produce univalent fragments after limited digestion with pepsin. The explanation is that papain cleaves on the amino-terminal side of the single disulfide bridge connecting the two heavy chains in rabbit IgG, whereas pepsin cleaves on the carboxyl-terminal side of the same bond, generating a single bivalent fragment, F(ab')2. Reduction of the interheavy chain bridge in the bivalent fragment yields univalent Fab'.
Nisonoff’s studies provided critical insights into the nature of the fragments produced by digestion with papain and their disposition in the intact antibody molecule. As noted by Julian Fleischman (3), the synthesis of data provided by the Porter laboratory on papain digestion and the Edelman and Porter groups on polypeptide chain separation into the four-chain model of the IgG molecule drew on Nisonoff’s experiments with pepsin digestion, which showed that the two fragments containing the active site (Fab or Fab') had to be located on the same side of the molecule, away from the Fc fragment. This effectively ruled out the previously popular cigar-shaped model in which the two active fragments were disposed on either side of a central Fc. Nisonoff’s work also clarified the nature of chromatographic fractions I and II obtained by Porter after papain digestion of rabbit antibodies. The similar yield initially found for fractions I and II was fortuitous, the result of charge heterogeneity in the antibody population and the choice of column conditions. In fact, the more negatively charged antibody molecules were found to contain two Fab of type I and the more positively charged two Fab of type II.
The F(ab')2 produced by pepsin retains the bivalence of the original antibody molecule and therefore the ability to precipitate or agglutinate antigen. However, it lacks the Fc fragment and therefore will not bind to cells expressing Fc receptors, eliminating much undesired "non-specific" antibody binding. The next logical step, taken by Nisonoff just as he was moving from Roswell Park to a position as Associate Professor of Microbiology at the University of Illinois, Urbana, was to show that the univalent Fab', generated by successive pepsin digestion and reduction, could be recombined into the bivalent F(ab')2 by oxidation, allowing the creation of bivalent antibodies of mixed specificity. Such hybrid antibodies have had many practical uses, for example, bringing a pharmacological agent into contact with a particular cell type.
Throughout his career, Nisonoff retained an interest in the three-dimensional structure of antibodies. In the late 1950s, this led to a collaboration with Cecil Hall and Henry Slater at Massachusetts Institute of Technology in an attempt to visualize the antibody molecule by electron microscopy. Although resolution was insufficient to discern the shape, the data provided a reasonable estimate of the size of the molecule. Methods of x-ray crystallography began to be applied to proteins in the 1960s and Nisonoff succeeded in obtaining crystals of Fab derived from human myeloma proteins. Preliminary structural work was conducted in collaboration with the group of Roberto Poljak, and this eventually led to a detailed understanding of the structure of the Fab, including localization of the active site and documentation of basic features of the Ig fold.
In 1966, Nisonoff moved from Urbana to the University of Illinois College of Medicine in Chicago, IL, where, in 1969, he assumed the Chair of the Department of Biological Chemistry. In Chicago, he continued a fruitful collaboration with Sheldon Dray relating structural features of rabbit antibodies to genetic variations known as allotypy. In work utilizing Nisonoff’s characteristic quantitative approach, they had shown that the population of IgG molecules in a rabbit heterozygous for allotype consists only of molecules displaying one or the other allotypic determinant, but not both. Coming on the heels of the proposal of the four-chain model for IgG, this finding suggested that the IgG molecule is symmetrical. Thinking that quantitative immunochemical methods would also be useful for investigating idiotypy (the unique antigenic specificity possessed by individual antibody molecules), Nisonoff embarked on a series of studies that laid the groundwork for widespread use of idiotypes as genetic markers.
During the 1960s immunologists were questioning the "one-gene-one-polypeptide" hypothesis because it was difficult to understand how the variable and constant regions of Ig heavy and light chains could be encoded by the same gene. By showing that IgG and IgM myeloma proteins obtained from the same patient had identical idiotypes, Nisonoff in collaboration with Hugh Fudenberg, provided convincing evidence that the V and C regions must be encoded by separate genes. In other studies, Nisonoff drew on his experience with antibodies directed against haptenic determinants, in this case arsonate, to address such questions as the size of the repertoire of antibody binding sites and the relationship of the idiotypic site of an antibody molecule to the Ag-binding site. He was among the first to show that all mice in a certain strain could be induced to express a shared, or so-called public idiotype. He postulated that these recurrent idiotypes represent germline genes. This idea was confirmed in a series of extensive amino acid sequence studies performed in collaboration with J. Donald Capra.
Nisonoff’s studies on shared idiotypes provided the means for tracking clonal lines of B lymphocytes, a technique widely used by many investigators to follow the migration and fate of Ag-specific B cells, laying the groundwork for studies of V gene usage and somatic mutation. Stimulated by earlier work by Dray and Rose Mage that expression of Ig allotypes could be suppressed by in vivo injection of anti-allotype antibodies, Nisonoff showed that injection of anti-idiotype antibodies resulted in long term suppression of idiotypes. This observation influenced Niels Jerne in formulating the idiotype network theory in which he proposed that the immune system could be regulated by anti-idiotype antibodies, which represent an internal image of Ag.
A major occupation and preoccupation of the later Chicago years was writing, together with John Hopper and Susan Spring, the monograph, The Antibody Molecule, a monumental annotated work providing a scholarly and comprehensive review of the structural studies (1). Choosing to review the field at this time was a reflection of Nisonoff’s astute insight, for it provided a definitive summary of the protein phase of molecular immunology and set the stage for the genetic era which was soon to follow. A decade later, Nisonoff wrote an introductory text of molecular immunology which showed not only his superb command of the subject, but also his skill in presenting complex material.
In 1975, Nisonoff moved to the Rosenstiel Research Center at Brandeis University. Interestingly, he was recruited by Harlyn Halvorson, then Director of the Rosenstiel Center, whose father, H. Orin Halvorson, had brought Nisonoff to the Microbiology Department at the University of Illinois, Urbana at the outset of his academic career. Intrigued by the idea that anti-idiotype antibodies may in some respect resemble Ag, Nisonoff proposed that anti-idiotype antibodies could be used as vaccines. Support was obtained by showing that mice immunized with antibodies directed against the idiotypic determinants of ant-viral antibodies were protected from infection with the same virus. Nisonoff’s views and thoughtful analysis of the idiotype field are cogently presented in his American Association of Immunologists Presidential Address (4).
Nisonoff’s extensive knowledge and clear intellect, as well as his commitment to fairness, made him much sought after as a member of editorial boards, panels, and committees. Heroically, he served three terms on the National Institutes of Health Allergy and Immunology study section, once as Chair. His most recent service, even after formal retirement from Brandeis, was on an National Institute of Allergy and Infectious Diseases Task Force, New Initiatives in Immunology, in which he played a major role in writing the final report. He became a member of the American Association of Immunologists in 1965 and served as its president in 1990–1991. He received many honors including membership in the National Academy of Sciences and the Pasteur Institute Medal.
To colleagues and students, as well as family and friends, Nisonoff was loyal and committed. He was as honest and straightforward in his human relationships as he was in his science. His characteristic modesty is encapsulated in the format of his curriculum vitae in which his honors are buried in a section captioned "other data." His sense of fun and good humor were legendary. He loved music and playing tennis. He had a strong lifelong sense of social justice and always took the side of the underdog. After retirement from Brandeis, he coached children in math and recent immigrants in English, and he was planning to take a course on teaching English as a second language. Without much fuss, he set about doing what he could to make his corner of the world a better place.
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References |
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TopIntroductionReferences |
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-globulin. Current Contents/Life Sci. 11:17.
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