ANNALS OF
`THE NEW YORK ACADEMY
`OF SCiENCES
`
`VOLUME 190
`
`
`
`NATION
`LIBRAR
`MAR :3 1972
`O.B'
`MEDICINE
`
`iham Kochwa
`em? G. Kunke!
`
`PUBLISHED BY THE NEW YORK ACADEMY OF SCIENCES
`
`1of18
`
`BI Exhibit1111
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`1 of 18
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`BI Exhibit 1111
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`

`

`::::i'IV&i~J~l~7.)~~:::~::::~;~.~~~;.~·~'l1~~~"!. ~~ ... -:~·l':;··::'.':"':"I:~~·~·.,~ .. ~;;!~:.,: :-o. ;;::· ;;;;i~:~;·::;;.'•::'"7:;;:;:;~-;;;~;;::i~~~;,-;;;~·=,~~;i~;;~~:::~:~·:_;;~~~)~:~·:;;:=;~;:::·~,·.,;.:~;·;~;;.~·~;\;,,~.~.~,:;/;·;~~~~;.~~~ -~,.:·.~~·:~:~;~_;·:~;;~~i~;:~·;.:-~-~~:~::.~: ~~~ .. ~~lw~~~~:;.~·~~-~~~~~~~-~:.;:~;:;;;~;;~~~~
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`... ), ... , .
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`
`,, .... . " .. :: ·., •. .
`
`,,.
`
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`
`• , .... "
`
`
`
`20f18
`
`BI Exhibit1111
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`2 of 18
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`BI Exhibit 1111
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`

`

`THE NEW YORK ACADEMY OF SCIENCES
`(Founded in 1817)
`BOARD OF GOVERNORS, 1971
`
`I. B. LASKOWITZ
`MARGARET MEAD
`Reccmfl11i: Secretary
`GEORGE I. I'UJIMOTO
`
`HERMAN COHEN
`
`PHILIP FEIGELSON
`
`M. JACK FRUMIN
`
`LLOYD MOT'.l
`J. JOSEPH LYNCH
`JACOB FELD
`Fi11a11cia/ Counselor
`FREDERICK A. ST AHL
`
`BERT N. LA DU, JR., Presidem
`SERGE A. KORFF, President-Elect
`
`Vice-Presidents
`
`Treasurer
`GORDON Y BILLARD
`Elected Govemors-at-Large
`1969-197/
`ROSS F. NIGRELLI
`1970-1972
`E. CUYLER HAMMOND
`1971-1973
`JOEL L. LEBOWITZ
`Part Presidents (Governors)
`
`IRVING J. SELlKOFF
`
`Executfre Director
`EDWIN S. SCHANZE
`
`DORIS PREGEL
`KENNETH W. THOMPSON
`Correspondi1111 Secretary
`H. CHRISTIN!! REILLY
`
`MAITHEW ROSENHAUS
`
`ETHEL TODACH
`
`SIDNEY A. SAVITI
`
`MINORU TSUTSUI
`HILARY KOPROWSKI
`N. HENRY MOSS
`Le11a1 Counselor
`EOWAlm D. BURNS
`
`ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
`
`Volume 190
`
`EDITORIAL STAFF
`
`Editorial Direclor
`PETER ALBERTSON
`
`Editor-in-Chief
`MARC KRAUSS
`
`Associate Ediwr
`MILDRED MONGE
`
`This conference was
`sponsored by The
`Academy's SECTION O F
`BIOLOGICAL AND
`MEDICAL SCIENCES
`Chairman
`LEONARD J. LERNER
`Vice-Chairman
`HAROLD YACOWITZ
`
`3 of 18
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`BI Exhibit 1111
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`ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
`
`VOLUMP. 190
`
`December 3 I, 1971
`
`IMMUNOGLOBULINS *
`
`Editors and Con/ erence Chairmen
`SHAUL °K OCHWA AND HENRY G. KUNKEL
`
`CONTENTS
`
`Part J. Structure of Jmm11noglobullns
`Antibody Structure and Molecular Immunology. By GERALD M. EDCLMAN . .
`Light Chain Structure and Theories of Antibody Diversity. By LEROY HooD,
`MICHAEL D. WATERFIELD, JAMES MORRIS AND CHARLES W. TODD ...... •
`Structure of JgA Proteins. By H . M. GREY, C. A. ABEL AND B. ZIMMllRMAN
`Structural Studies of Immunoglobulin E: I. Physicochemical Studies of JgE
`Molecule. By S. KOCHWA, w. D. TERRY, J. D . CAPRA AND N. L. YANG
`Structural Studies of Immunoglobulin G, M and A Heavy Chains. By
`B. FRANGIONE, F. PRELLI, C. MlllAESCO, C. WOLFENSTEIN, E. MlllAESCO
`AND E. c. FRANKLIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`The Amino Acid Sequence of Human Macroglobulins. By F. W. PUTNAM,
`A. SurMtzu, C. PAUL, T. S1UNODA AND H. KOHLER . . . . . . . . . . . . . . . . . . .
`The Three-Dimensional Conformation of "YM and "YA Globulin Molecules. By
`A. FEINSTEIN, E. A. MUNN AND N. E. RICHARDSON . . . . . • . . . . . . . . . . . .
`X-Ray Diffraction and Electron Microscope Studies on a Crystalline Human
`Immunoglobulin. By D . R. DAVIES, R. SARMA, L. W. LADAW, E. Sn,veR-
`TON, D . SUGAL AND W. D. TERRY .. .. .... , . . . . . . . . . . . . . . . . . . . . . . . .
`
`Part D. GeoeCics of lmmunoglobulins
`
`Immunoglobulin Genetics in Cellular Immunology. By LEONARD A. HER.Zl!N·
`BERO
`...••. , , .•.••..••.•..•.•• , . . . . • . . • . . • • . . . • . • . • • • • • • • • • • . •
`Formal Genetics of the Immunoglobulin Systems. By ERNA VAN LOOllCM....
`Genetic Control of the Constant Homology Regions of Immunoglobulin G
`Heavy Chain Subclasses. By J. JJ. NATVIG, M. W. TuRNf!R AND T. E.
`MICllAtlLSEN
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Genetic Control of Rabbit H Chain Biosynthesis. By JAMllS W. PRAnL AND
`C11Ant.f!S W. TODD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Genetic Control of Specific Immune Responses in Inbred Mice. By F. CARL
`GRUMET, GRAHAM F. MITCHELL AND HUGH 0. MCDEVITT . . . . . . . . . . . .
`
`5
`
`26
`37
`
`49
`
`71
`
`83
`
`104
`
`122
`
`130
`136
`
`150
`
`161
`
`170
`
`* This series of papers is the result of :l conference entitled Immunoglobulins, held by
`The New York Academy of Sciences on March 8-10, 1971.
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`{_1' ·~~ 1, J .
`r1 I :, ·
`
`Part Ill. Synthesis of lmmunoglobulins
`
`Introduction to the Session on Biological and Cellular Aspects of the Regula-
`tion of Immunoglobulin Synthesis. By llAltU.r BcNACERRAF . . . . . . . . . . . .
`Effect of Antigenic Structure on Antibody Biosynthesis. By MICHAEL SELA. . .
`Normal and Altered Quantitative Expressions of Allotypes on Light and
`Heavy Chains of Rabbit Immunoglobulins. By RosE G. MAGE . . . . . . . .
`Chronic Allotype Suppression in Mice: An Active Regulatory Process. By
`LEONORE A. H1mzEN1JERG, E. B. JAconsoN, LEONAiu.> A. HERZENIJERG
`AND ROY J. RIBLET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Proliferation and Differentiation of Lymphoid Cells: Studies with Human
`Lymphoid Cell Lines and Immunoglobulin Synthesis. By JOHN L. FAHEY,
`DONALD N. BUELL AND HAROLD c. Sox . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`The Regulation of Jmmunoglobulin Synthesis and Assembly. By R. BAUMAL,
`P. CoFFINo, A. BARGilLLESJ, J. BUXBAUM, R. LAsKov ANU M. D. SCHARF!'
`Synthesis and Intracellular Transport of Immunoglobulin in Secretory and
`Nonsecretory Cells. By CHARLES J. SullRH, Js,uc Sc1mNKEJN AND
`JoNATHAN w. UHR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Antibodies Induced by Local Antigenic Stimulation of Mucosa! Surfaces. Dy
`J. F. HEREMANS AND H. BAZIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`l'art IV. Monoclonal Antibodies
`
`Introduction to Session on Homogeneous Antibodies. By RICHARD M. KRAUSE
`Homogeneous Elicited Antibodies: Induction, Characterization, Isolation, and
`Structure. By EDGAR HAnER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Antigen-Binding Myeloma Proteins in Mice. Dy MrcHAEL PO'ITER . . . . . . . . .
`Use of Homogeneous Immunoglobulins for Studying the Effects of Antibody
`Polyvalence. By H ENRY METZGER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Cold Agglutinins: Studies of Primary Structure, Serologic Activity and Anti-
`genic Uniqueness. By RALPH c. WILLIAMS, JR. . . . . . . . . . . . . . . . . . . . . . .
`
`Part V. Active Sites of Antibodies
`
`Affinity Labeling of the Active Sites of Antibodies and Myeloma Proteins. Dy
`S. 1. SINGER, NANCY MARTIN AND NEAL 0. THORl'c . . . . . . . . . . . . . . . . . .
`Combining Sites of Immunoglobulins that Bind the 2,4-Dinitrophenyl (DNP)
`Group Specifically. Dy JosEPH HAIMOVICH, HERMANN. EISEN AND DAVID
`GI VOL
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Individually Specific Antigenic Determinants Shared by a Myeloma Protein
`and Nonspecific IgG. By STEl'HEN K. WILSON, Birnell W. BRlENT AND
`ALFREI> N!SONOFF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Structure-Function Relationships Among Anti-Gamma Globulin Antibodies.
`By J. DONALD CAPUA, J. MICHAEL KEHOE, RODERT J. WINCHCSTER AND
`HENRY G. KUNKEL
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Attempts to Locate Complementarity-Determining Residues in the Variable
`Positions of Light and Heavy Chains. By ELVIN A. J(,\DAT AND T. T. WlJ
`Mapping the Combining Sites of Antibodies Specific to Polyalanine Chains.
`/Jy ISRAEL SCHECHTER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Immunoglobuiins as Cell Receptors. By BENEVENUTO PERNIS, LUCIANA FoRNI
`AND LUISA AMANTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Chemical Approaches to the Cell Receptor Problem. By LEON Wor-SY,
`P.AOLO TRUFFA-BACHl AND DAVID NAOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`178
`181
`
`203
`
`212
`
`221
`
`235
`
`250
`
`268
`
`276
`
`285
`306
`
`322
`
`330
`
`342
`
`352
`
`362
`
`371
`
`382
`
`394
`
`420
`
`432
`
`Copyri_ght, 1972, by The New York Academy of Sciences. All rights reserved. Except
`for brief q11otatio11s by reviewers, reproduction of this p11b/icatio11 in whole or i11 part by
`a11y means whatever is strictly prohibited without written permission from the rmb/isher.
`
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`Part VJ.
`
`lmmunoglobulins in Disease
`
`IgE and Reaginic Hypersensitivity. By KIMISHIGE TSlllZAKA AND TERUKO
`JSlllZAKA
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Heavy Chain Diseases. By EDWARD c. FHANKLIN, BLAS FRANGIONE AND
`S1HlLDON COOPER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Structural Studies of Gamma Heavy Chain Disease Proteins. Dy Wn.LIA.M D .
`TERRY AND DANIEL EIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`A Crystalline -y"' Human Monoclonal Protein with an Extensive H C hain
`Deletion. By H. F. DEUTSCH AND T. SuzuKr . . . . . . . . . . . . . . . . . . . . . . . .
`Studies on Alpha Chain Disease. By MAXIMll Sl!LIGMANN, EDITH MIHAnsco
`AND BLAS FRANGJON!l
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Studies of An Unusual Biclonal Gammopathy: Implications with Regard to
`Genetic Control of Normal l mmunoglobulin Synthesis. By H. H. FuDl!N-
`DEllG, A-C. WANG, J. R. L. PINK AND A. s. LEVIN . . . . . . . . . . . . . . . . . . . .
`Pathologic Conditions Associated with Plasma Cell Dyscrasias : A Study of
`806 Cases. By TAKASHI Jso1m AND ELLIOTI F. OSSERMAN . . . . . . . . . . . . .
`Jrnmunochemistry of the Rh System: V. Determination of Rh Agglutinating
`Activity and JgG Content of Sequential Eluates for the Assay of Rh
`Antibody. By }{1CHARD E. ROSENFIELD, EUGENE M. BEI\KMAN, JACOB
`NUSDACHER, CHARLO"JTI! DADINSKY AND SHAUL Koc11WA . . . . . . . . . . . . . .
`
`443
`
`457
`
`467
`
`472
`
`487
`
`50 1
`
`507
`
`519
`
`Summary: The Take-Home Lesson-1971. By MELVIN COHN . . . . . . . . . . . .
`
`529
`
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`

`A TIEMPTS TO LOCATE COMPLEMENTARITY(cid:173)
`DETERMINING RESIDUES IN THE VARIABLE
`POSITIONS OF LIGHT AND HEAVY CHAINS *
`
`Elvin A. Kabat and T. T. Wu
`
`Departments of Microbiology, Neurology, & Human Genetics & Development,
`Columbia University, The Neurological Institute, Presbyterian Hospital
`New York, N.Y.
`and Departments of Physics and Engineering Sciences
`Northwestern University
`Evanston, Ill.
`
`Examination of the sequences of Bence-Jones proteins and myeloma immu(cid:173)
`noglobulin light chains from humans and from mice has led to the recognition
`of the variable and constant regions, and the accumulation of data on heavy
`chains indicates that they too contain a variable and a constant region. 1 -0 The
`variable region comprises approximately the amino terminal half of the light
`chain and the amino terminal quarter of the heavy chain, and it is these regions
`which are generally believed to be responsible for antibody complementarity.
`The genetic control of the constant regions of both chains is readily explain(cid:173)
`able on classical genetic principles, but the genetics of the variable regions is
`far from clear and no generally agreed upon concept of the genetic determina(cid:173)
`tion of antibody complementarity has as yet been formulated. The recognition
`of subgroups in the variable regions of human (Reference 2, p. 133),6 • 7 and
`mouse 8 light chains and in human heavy chains, 9 • 11 from sequence analyses,
`mainly of the first 20-25 amino terminal residues, has led to the designation of
`genes for these subgroups. It is generally accepted that the light chain and the
`heavy chain are each under the control of two genes, one for the variable and
`one for the constant region, and that a translocation results in the joining of
`these two genes. 12 These conclusions, however, do not account for antibody
`complementarity nor do they localize the combining site to any specific portions
`of the variable region.
`When the subgroups of the variable regions were first recognized, it was
`noted 7• 4
`that certain positions, notably those near 30 and 91-96, showed
`greater variability than could be accounted for by the subgroups. As further
`sequences accumulated, it became clearer that there were two regions of hyper(cid:173)
`variability, one following cysteine 23 and the other following cysteine 88 and
`comprising residues 24-34 and 89-96 respectively, and it was of special inter(cid:173)
`est that these two regions were brought into close proximity by the disulfide
`bond 123-1188 , and that insertions or deletions occurred in these regions. 13 • 4
`A more detailed analysis indicated the presence of three hypervariable
`regions. Franek (Reference 5, p. 311 ) tabulated the positions showing non(cid:173)
`homologous replacements and recognized a region from residues 50-55 in
`
`*Aided by grants from the National Science Foundation, GB-8341 and GB-25686,
`a General Research Support Grant from the United States Public Health Service
`to Columbia University, and Biomedical Sciences Support grant FR 7028-03 from the
`National Institutes of Health to Northwestern University.
`
`382
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`Kabat & Wu: Residues of Light and Heavy Chains
`
`383
`
`addition to the other two regions. A statistical analysis was made by Wu and
`Kabat 14 •" of the complete and partial sequence data available on 77 Bence(cid:173)
`Jones proteins and immunoglobulin light chains, considering human K, human
`,\, and mouse K chains of various subgroups as a single population which was
`aligned for maximum homology. Defining variability as
`
`Number of different amino acids at a given position
`Frequency of the most common amino acid at that position '
`
`three hypervariable regions were found involving residues 24-34, 50--56 and
`89-97. It was proposed that at least the first and third of these regions, and
`possibly all three, together with similar regions in the heavy chains might be
`the complementarity determining regions and that amino acid side chains in
`these regions might make contact with the antigenic determinant, the remain(cid:173)
`der of the residues of the variable region being essentially structural and in(cid:173)
`volved in three-dimensional folding.
`The significance of the three hypervariable regions was greatly reinforced
`by the findings of Weigert and coworkers 15 who examined ten mouse ,\ chains;
`from composition analyses on peptides, six had apparently identical sequences
`in the variable regions and the remaining four showed variation only in one
`or another of three hypervariable regions. FIGURE 1 shows the sequences of
`Weigert and associates superimposed on the original plot of Wu and Kabat.
`
`L~ur:ibcr of
`Examples
`
`l
`l
`
`32
`25
`ER-SER
`
`ASN-SER
`
`ER-SER
`
`..JIB~LY
`
`_N-SER
`
`Position
`
`2
`50
`IlE-GLY
`
`ILE-GLY
`
`....fil!.-GLY
`
`ILE-GLY
`
`ILE-~
`
`97
`
`112
`
`2;
`
`50
`Position
`;>.. myeloma sequences of Weigert and coworkers 10 super(cid:173)
`FIGURE I. Mouse
`imposed on the variability against position plot for human K, human ;>.. and mouse K
`Bence-Jones proteins and light chains of Wu and Kabat.14 The positions at which no
`differences among the mouse ;>.. chains were found are indicated by a line. Sequences
`are given at positions at which differences occurred, and the number of nucleotide
`changes indicated by the underlining.is
`
`75
`
`I
`100
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`384
`
`Annals New York Academy of Sciences
`
`It should be noted that their positions 50 and 52, if aligned for homology
`relative to our data, would be at 48 and 50. The mouse ,\ chains thus seem to
`be much more homogeneous in the variable region than mouse K or human K
`or ,\ chains, so that the changes appear to be restricted to the hypervariable
`regions.
`It is also noteworthy that Capra and Kunkel 16 have found two cases of
`hypergammaglobulinemic purpura with antibodies of restricted specificity to
`have identical sequences for the first 40 N-terminal residues of their light
`chains that include the first hypervariable region; both chains were unusual in
`having Thr-Val at residues 13 and 14. It would be of great interest to estab(cid:173)
`lish whether the combining sites are identical and whether the sequence of the
`entire variable region of the light chains (and ultimately of the heavy chains)
`proves to be identical. Wang and colleagues 17 • 18 have shown that in a patient
`with both a myeloma yG 2K and a macroglobulin y MK protein, the K light chains
`of both were identical in amino aci<;! composition, bands in urea-starch gel, pep(cid:173)
`tide maps, and in optical rotatory dispersion and circular dichroism. The light
`chains have not been sequenced, but the first 27 residues from the N-terminal
`region of the heavy chains and the idiotypic specificity of both proteins were
`identical.
`We have continued to tabulate sequences of light chains and have now
`accumulated complete and partial sequences data on 121 human K and ,\ and
`mouse K and ,\ sequences. The tabulation only includes data for positions at
`which the sequence is reported as unequivocal. FIGURE 2 shows the variability
`vs. position plots for all 121 light chains as a single population (A), for 33
`human Kl chains (B), for 58 human K chains (C), and for 58 human and 27
`mouse K chains (D). It is evident that the hypervariable regions may be seen
`in all plots. When two values are given for any position this is due to uncer(cid:173)
`tainty in regard to Glx and Asx residues. It should be emphasized that apart
`from the N terminus, the number of complete sequences is still not as large as
`desirable and that the data for positions 40-85 are based on less than 20 pro(cid:173)
`teins, while those at positions 1-23 are based on between 59 and 117 proteins.
`It is of considerable interest that Singer and Thorpe found the invariant
`Tyr 86 of the light chain to be affinity-labeled in anti-DNP antibodies/" and
`Goetz) and Metzger 26 showed that position 34 in the above alignment (actu(cid:173)
`ally position 32) in the mouse ,\ myeloma protein with anti-DNP activity was
`labeled. Tyr 86 is very close to the hypervariable region, and position 34 is
`in the first hypervariable region. The identification of residues by affinity
`labeling in antibodies and myeloma proteins with different specificities will
`obviously be of importance in defining the relation of residues in the hyper(cid:173)
`variable regions to antibody complementarity.
`Sequence data on heavy chains have been accumulating, and we have
`examined these data for hypervariable regions. Complete sequences of seven
`heavy chain variable regions, including five yGl (Eu, He, Daw, Cor, Nie), 1
`yM (Ou), one mouse myeloma protein (MOPC 173), and partial sequences
`of 7 yGl, 2 yG2, 2 yG3, 1 yG4, 8 yM, 2 yA, 1 yE, four rabbit, two horse and
`one shark, were available. As for light chains, subgroups of the variable
`regions of the heavy chains have been recognized and termed Vm, Vm 1, V11m,
`and Ymv· 9 • 20 • 10 • 11 The degree of homology of these variable region sequences
`is truly extraordinary. FIGURE 3A shows a plot of variability against position
`considering all the heavy chain sequences available as a single population.
`In aligning for maximum homology, gaps of two residues are placed between
`
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`

`Kabat & Wu: Residues of Light and Heavy Chains
`150 c
`
`150 A
`
`385
`
`HUMAN KAPPA,HUMAN LAMBDA
`MOUSE KAPPA,MOUSE LAMBDA
`
`HUMAN KAPPA
`
`100
`
`GAP
`
`•
`
`GAP GAP
`
`• • 100
`
`GAP •
`
`GAP GAP • •
`
`BO B
`
`HUMAN KAPPA I
`
`BO D
`
`HUMAN KAPPA
`MOUSE KAPPA
`
`40
`
`40
`
`POSITION
`FIGURE 2. Variability at different amino acid positions for the variable region
`of light chains. GAP indicates positions at which insertions have been found. Data
`used: Wu and Kabat," plus the following light chains: Pot, Die, Car, Tei, Joh (16);
`Dav, Fin (19); Ou (20); Hau (21); Ti!, Wil, Sal, Porn (22); Ful (23); G 173,
`F 31C, BJ 149, BJ 321, BJ 63, A 603, A 870, A 384, A 467, F 47, H 37, BJ 843,
`B J 674, A 8, A 15, B J 773, BJ 265, GLP C 1, A 167 (24); S 104, X P 8, J 698,
`H 2061, J 558, HOPC 1, RPC 20, S 176, H 2020, S 178 (15).
`
`residues 34 and 35 of Vm, V11m, and mouse proteins, of one residue between
`positions 54 and 55, of three residues between positions 85 and 86 in some
`V 11u proteins, and of three to five residues between positions 100 and 101
`in certain V HI• V 11u and V 11111 proteins. Hypervariable regions are seen as
`for the light chains. The first hypervariable region comprises residues 31-35,
`and the last hypervariable region involves residues 95-102; in both instances,
`as with the light chains, gaps occur in both regions. There also are indications
`of two other regions which are somewhat more variable. One of these runs
`from residues 50--65 and corresponds approximately to the third light chain
`hypervariable region but is considerably larger; and the fourth, which was
`noted by Ors. Capra and Keogh, includes residues 81 and 83-85. A similar
`distribution is seen in FIGURE 38, in which only the human heavy chain
`sequences are plotted; in this instance the hypervariable region extends from
`50--54 rather than from 50--65, although somewhat more variability is seen
`for residues 61, 62, and 64, and residues 81, 83, 84, and 85 also show some-
`
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`386
`
`Annals New York Academy of Sciences
`
`HUMAN, MOUSE, RABBIT, HORSE,
`SHARK HEAVY CHAINS
`
`GAP GAP
`
`• •
`
`GAP GAP
`
`• •
`
`25
`
`50
`
`75
`
`100
`
`HUMAN HEAVY CHAINS
`
`00 A
`
`40
`
`>(cid:173)...
`
`..J
`CD
`<I:
`~ 00 B
`
`40
`
`25
`
`50
`
`75
`
`100
`
`POSITION
`FIGURE 3. Variability at different amino acid positions for the variable regions of
`heavy chains. GAP indicates the positions at which insertions have been placed.
`Data used: Eu;21 Ca;zs Ste, horse yGab, horse yGT;W Dee; 3 0 He;o Daw, Cor; 31
`Ou ;3o Car ;:n Sa;H Vin;35 Til;•s Sha;JG Nie;" Tei, Was, Jon, Ben;0 2 Fi, Vu ;3; Zuc;3s
`Bus, Dau, Dos, BaJ;a9 Di, Wo, Na, Hu, Re;to MOPC 173;40 Rabbit;41 Rabbit Aal,
`Aa2, Aa3 ;4 2 Shark.43
`
`what increased variability. Residue 5 in both plots shows substantial variability.
`The two major hypervariable regions are clearly evident. Unlike the light chain
`hypervariable regions, which begin after each Cys residue, the heavy chain
`hypervariability begins at residue 31 and 95 respectively, while the Cys residues
`are at positions 22 and 92. Thus while they too are brought into close prox(cid:173)
`imity by the disulfide bond, there appears to be some displacement from the
`Cys residues.
`
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`

`Kabat & Wu: Residues of Light and Heavy Chains
`
`387
`
`The symmetry in the location of hypervariable regions in the light and
`heavy chains which are brought into relatively close proximity by the respec(cid:173)
`tive intrachain disulfide bonds can provide a three-dimensional site which could
`contain the complementarity determining residues, e.g., those which make con(cid:173)
`tact with the antigenic determinant. As with the light chains, deletions or
`insertions occur in these two hypervariable regions. The role of the other
`hypcrvariable region is not clear. It is possible that it too is involved in site
`complementarity or, alternatively, it could contribute to the specificity of
`recognition of heavy and light chains. Its role will be more clearly established
`when some idea of its relation to the other hypervariable regions in three
`dimensions emerges. More sequence data on heavy chains are obviously needed.
`The hypervariability at positions 81 and 83-85, if substantial when more
`sequences are available, might also be related to site complementarity.
`
`Species-Specific Residues
`
`In 1967, when only a few human Kand two mouse K Bence-Jones proteins
`had been sequenced, a comparison of the variable and constant regions showed
`that although mouse K differed from human K in the constant region at 43
`positions, in the variable region there were many fewer positions, only about
`I 0-14, at which the amino acids found in the mouse chains differed from
`those in the human chain. 41 This indicated a substantial deficit of species(cid:173)
`specific residues in the variable region, and indicated substantial selective pres(cid:173)
`sures in evolution to preserve the set of variable sequences. Subsequent analy(cid:173)
`ses 1 ~. ' ·1 of all the human K, human ,\, and mouse K chains as a single popula(cid:173)
`tion reduced the number of species-specific residues to 2 out of the I 07 in the
`variable region. It should be emphasized that this definition of species-specific
`residues is based essentially on structural considerations, i.e., do Bence-Jones
`proteins exist in either human or mouse with amino acids which differ at a
`given position in the variable region or, conversely, to form a functional chain
`is it necessary to restrict the amino acids present to such an extent that the
`human and mouse can have only the same limited set of amino acids at a
`given position?
`The discovery of subgroups of K and of ,\ chains have led other workers
`to consider that more residues in the variable region were species-specific.
`However, although the number of species-specific residues of the variable
`region may be questioned, there is little doubt that there are many fewer in
`the variable region than the 43 in the constant region.44 • 13 • 14
`We have reexamined the question of species specificity, since many more
`sequences are now available. Unfortunately, however, there are still only two
`complete mouse K sequences. TABLE I summarizes the results. Considering
`all human K and ,\ and mouse K proteins, there are but two positions, 50 and
`96, in the variable region at which the mouse differs from the human . Position
`96 is the most hypervariable position thus far reported, and it is doubtful
`whether it can be considered species-specific; the constant region has 36 spe(cid:173)
`cies-specific positions computed on this basis. If one compares only human K
`with mouse K, there are 9 species-specific positions in the variable and 43 in the
`constant region. Comparing human Ki with mouse K1,
`the species-specific
`residues are 24 and 43 respectively.
`
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`Annals New York Academy of Sciences
`
`TABLE 1
`
`SPECIFS-SPECIFIC RESIDUES IN THE CONSTANT AND VARIABLE REGIONS OF HUMAN AND
`MOUSE BENCE-JONES PROTEINS AND IMMUNOGLOBULIN LIGHT CHAINS*
`
`Proteins Compared
`
`Variable Region
`
`Constant Region
`
`Species-Specific Residues
`
`Human IC, human X, mouse IC
`Human IC, mouse I(
`
`Human 1C1, mouse ICJ
`
`2/ 107 (positions 50, 96)
`9/107 (positions 36, 43, 55, 60, 72,
`80, 85, 92, 96)
`24/ 107 (positions 26, 30, 32, 33, 36,
`39, 42, 43, 44, 46, 51, 55, 60, 66, 69,
`71, 72, 80, 84, 85, 89, 92, 94, 96)
`
`36/ 107
`43/107
`
`43/ 107
`
`• Only one mouse 1C1 and two mouse IC Bence-Jones proteins have been sequenced beyond
`residue 24.
`
`The last two comparisons are of special interest since beyond residue 24,
`only two mouse K and only one mouse Kr proteins have been sequenced. It
`is of interest that no species-specific positions occur in the first 24 residues
`for which a number of mouse K and Kr proteins have been sequenced. Thus,
`the species-specific positions in TABLE l represent the maximum number, and
`this number can only be reduced as additional mouse sequences become avail(cid:173)
`able. However, even for the Ki proteins, the lack of species-specific residues in
`the variable as compared with the constant region is highly significant. This
`becomes self-evident when one considers that the problem of alignment of
`variable regions to establish maximum homology is much simpler than with
`most other proteins of similar size and can be readily accomplished by inspec(cid:173)
`tion. Indeed, the similarity in sequences of the variable region of the heavy
`chains of different species suggests that very few residues of the heavy chain
`will show species specificity.
`
`Role of Glycine
`
`The unique pattern of invariant glycines in the variable regions of light
`chains of immunoglobulins and Bence-Jones proteins led to the suggestion that
`they might be responsible for conferring flexibility on the chains to permit
`three-dimensional folding to accommodate the numerous substitutions which
`were observed at the variable positions.45 • 13• 14 Consideration of the cf> and if
`angles which glycine exhibited in other known proteins indicated that it had a
`much wider range than was seen for all other amino acids, so that it could
`indeed be responsible for conferring flexibility. 14 The unusual distribution of
`invariant glycines at positions 99 and l 01 which-with the frequently occur(cid:173)
`ring Gly at position 100 to give the sequences Gly-Gln-Gly or Gly-Gly-Gly
`near the end of the variable region and close to the two hypervariable regions
`joined by the disulfide bond 123-11"8-led to the suggestion that these glycines
`might serve as a pivot to facilitate optimal contact of the complementarity
`
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`

`Kabat & Wu: Residues of Light and Heavy Chains
`
`389
`
`determining residues with the antigenic determinant 45 (Reference 2, p. 87). It
`was also noted that two glycines were present at an analogous position in all
`heavy chains 14 in a sequence Gly-Gln-Gly, Gly-Arg-Gly or Gly-Gly, and thus
`might also function as a pivot.
`It is well established that the walls of the
`lysozyme cleft move slightly to make optimal contact with its substrate.4 6 The
`importance of good contact for binding of an antigenic determinant in the
`antibody combining site would tend to make such a pivot type of structure
`favored evolutionarily.
`TABLE 2 summarizes data on the occurrence of glycine in the heavy chains
`for those complete human sequences which are available as well as for partial
`sequences of human, mouse, rabbit, and shark immunoglobulins. Despite the
`few sequences available, the data fall into three groups with respect to the fre(cid:173)
`quency of occurrence of glycines comparable to those found for the light
`chains:
`A (80-100%): positions 8, 26, 42, 104, and 106. These may prove to be
`largely invariant glycines. Position I 04 is not glycine in
`Eu but, as noted, there is a second glycine in position
`I 07 that together with the glycine at 106 was hypothe(cid:173)
`sized to function as a pivot. Their exact position is
`affected by the insertion of gaps in aligning for maxi(cid:173)
`mum homology.
`
`TABLE 2
`FREQUENCY OF OCCURRENCE OF GLYCINE RESIDUES AT VARIOUS POSITIONS IN THE
`VARIABLE REGIONS OF HEAVY CHAINS
`
`No. of Glycines
`No. of Proteins
`Sequenced at
`that Position
`
`Position
`
`Percent
`Glycine
`
`Position
`
`No. of Glycines
`No. of Proteins
`Sequenced at
`that Position
`
`Percent
`Glycine
`
`6
`8
`9
`10
`13
`15
`16
`17
`26
`27
`29
`31
`33
`35
`40
`42
`44
`
`1/22
`21/22
`14/22
`10/22
`1/22
`16/ 21
`10/ 20
`1/ 19
`18/ 18
`2/ 17
`1/ 15
`1/ 15
`2/ 14
`2/11
`1/7
`7/7
`4/7
`
`5
`96
`64
`45
`5
`76
`50
`5
`100
`12
`7
`7
`14
`18
`14
`100
`57
`
`49
`50
`55
`60
`65
`85A
`85C
`94
`95
`96
`97
`IOOC
`104
`106
`107
`
`2/ 7
`1/7
`1/7
`1/7
`2/7
`1/7
`1/ 8
`2/ 10
`1/ 8
`1/8
`2/ 8
`1/ 1
`4/5
`5/ 5
`1/5
`
`29
`14
`14
`14
`29
`14
`13
`20
`13
`13
`25
`100
`80.
`100
`20 •
`
`•The Gly at position 107 occurs in protein Eu, which lacks Gly at 104, thus all proteins
`have the sequence Gly-Gln-Gly, Gly-Arg-Gly or Gly Gly at positions 104, 105, 106, or
`106, 107.
`
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`C (5-29%):
`
`B (50-76%) : positions 9, 10, 15, 16, 44. These glycines might also
`contribute to flexibility; although the number of sequen(cid:173)
`ces is limited, they appear to be specific for one or
`another of the variable region subgroups.
`positions 6, 17, 27, 31, 33, 35, 40, 49, 50, 55, 60, 65,
`85A, 85C, 94, 95, 96, 97. These glycines might be
`involved in specificity of variable region subgroups or
`in site complementarity, or perhaps the total number of
`sequences available is too small to establish their role .
`The total number of invariant glycines (A) in the variable region is some(cid:173)
`what lower for the heavy chains than for the light chains for which seven
`invariant glycines were found. The glycines in group B seem to be invariant
`in one or more of the heavy chain variable region su