THE COVALENT STRUCTURE OF
`AN ENTIRE -yG IMMUNOGWBULIN MOLECULE*
`
`BY GERALD M. EDELMAN, BRUCE A. CUNNINGHAM, w. EINAR GALL,
`PAUL D. GO'ITLIEB, URS RUTISHAUSER, AND MYRON J. WAXDAL
`
`THE ROCKEFELLER UNIVERSITY
`
`Communicated by Theodore SMdlovsky, March 21, 1969
`
`Abstract.-The complete amino acid sequence of a human -yGl immunoglobu­
`lin (Eu) has been determined and the arrangement of all of the disulfide bonds
`has been established. Comparison of the sequence with that of another myeloma
`protein (He) suggests that the variable regions of heavy and light chains are
`homologous and similar in length. The constant portion of the heavy chain con­
`tains three homology regions each of which is similar in size and homologous to
`the constant region of the light chain. Each variable region and each constant
`homology region contains one intra.chain disulfide bond. The half-cystines
`participating in the interchain bonds are all clustered within a stretch of ten
`residues at the middle of the heavy chains.
`These data support the hypothesis that immunoglobulins evolved by gene
`duplication after early divergence of V genes, which specified antigen-binding
`functions, and C genes, which specified other functions of antibody molecules.
`Each polypeptide chain may therefore be specified by two genes, V and C, which
`are fused to form a single gene (translocation hypothesis). The internal homol­
`ogies and symmetry of the molecule suggest that homology regions may have
`similar three-dimensional structures each consisting of a compact domain which
`contributes to at least one active site (domain hypothesis). Both hypotheses
`are in accord with the linear regional differentiation of function in antibody
`molecules.
`
`Antibodies or immunoglobulins can interact with a wide range of different
`antigenic determinants and, after specific binding to an antigen, they play a
`fundamental part in physiological functions of the immune response. The
`specificity of antigen binding depends ultimately upon amino acid sequences of
`the variable or V regions of antibody molecules. It is the diversity of these
`sequences which results in the range of specificities required for a selective im­
`mune response. In contrast, other regions of the antibody molecule have rela­
`tively constant sequences and are responsible for physiological functions. Like
`enzymes, these C regions appear to have evolved for a restricted set of interac­
`tions. This unusual picture of intramolecular differentiation has emerged from
`studies of the structure of immunoglobulins from different animal species. I To
`date, only portions of immunoglobulin molecules have been suhjected to amino
`acid sequence determination.
`We now report the amino acid 8equeuce of an entire human -yGl immuno­
`globulin (molecular weight 150,000), the location of all disulfide bonds, the
`arrangement of light and heavy chains, and the length of the heavy chain V
`region.
`
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`
`MaterWls and Metlw<U.-The isolation of the myeloma protein Eu2 and the preparation
`of its CNBr fragment53• 4 have he.en described. Similar methods were used for the isola­
`tion of the -yG 1 myeloma protein He and for the preparat.ion of its CNBr fragments.
`We have previously described the methods used for enzymatic digestion with trypsin,
`chymotrypsin, and pepsin, gel filtmtion, ion exchange chromatography, high voltage paper
`electrophoresis, determination of NHrterminal and COOR-terminal residues, amino acid
`analysis, and determination of amino acid sequences by the dansyl-Edman procedure. •-e
`The positions of glutamine and asparagine were assigned9 by determining the electro­
`phoretic mobility of peptides and by amino acid analysis of the peptides after enzymatic
`hydrolysis. The half-eystinyl residues contributing to each intrachain disulfide bond
`were determined 10 using the diagonal electrophoresis method.11
`Results.-The organization of the whole molecule is shown in Figure 1; an un­
`equivocal proof of the arrangement of the two identical light chains and two
`identical heavy chains has already been given.4 Each light chain is linked to its
`neighboring heavy chain by a disulfide bond between corresponding half-
`
`Fob(t)
`
`Fob(t)
`
`Heovy
`choin
`
`Light
`choon
`
`COOi<
`COOH
`Fc(t)
`
`FIG. 1.---0ver-all arrangement of chains and disulfide bonds of "YGl immunoglobulin
`Eu. Half-cystinyl residues are numbered I-XI; numbers I-V designate corresponding
`residues in light and heavy chains. PCA: pyrollidooecarboxylic acid. CHO: carbo­
`hydrate. "Fab(t)" and "Fc(t)" refer to fragments produced by trypsin, which cleaves
`the heavy chain as indicated by dashed lines above half-cystinyl residues VI. VH, VL:
`variable regions of heavy and light chains, CL: constant region of light chain. CHl,
`Ca2, Ca3: homology regions comprising Ca or constant region of heavy chain.
`
`cystines V. Half-cystines VI and VII form bonds linking the half-molecules via
`the heavy chains. Trypsin cleaves the molecule at lysyl residue 222 to form two
`Fab(t) and one Fc(t) fragments.2• 5
`There are several strikingly linear arrangements in the primary structure.
`From their amino termini to half-cystines V, the light and heavy chains can be
`aligned or put in register. The intrachain disulfide bonds are linearly and period­
`ically disposed.12• 13 In accord with the alignment of light and heavy chains,
`corresponding intrachain disulfide bonds are in similar positions and the disulfide
`loops are of approximately the same size.
`Previous studies7 have suggested that V regions of light and heavy chains have
`similar lengths and begin at the NHrtermini; this will be confirmed below.
`The CL region of the light chain has the same length as V L, but the CH region of
`the heavy chain is about three times as long. CH may be divided into three
`
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`80
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`BIOCHEMISTRY: EDELMAN ET AL.
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`PROC. N. A. s.
`
`homologous regions of approximately equal length: Cul, C82, and C83 (Fig. 1).
`We have already reported the amino acid sequence of the first 877 and the last
`224 residues8 of the heavy chain as well as the partial sequence of the entire light
`chain.6 The complete amino acid sequence of the light chain (214 residues) is
`shown in Figure 2. Positions of the half-cystiuyl residues may be compared
`with Figure 1 and the methionyl residues may be correlated with previous studies
`on the CNBr fragments of Eu.3• 4 The variable region extends through residue
`108. In accord with other studies,1 valine 191 is related to the Inv specificity. 2
`The complete sequence of the heavy chain (446 residues) is presented in Figure
`3 which may be compared with Figure 2 for alignment with the light chain
`sequence. Isolation of a single glycopeptide8 indicated that the polysaccharide
`
`10
`20
`l
`ASP-I LE-GLN-MET-THR-GLN-SER-PRO-SER-THR-LEU-SER-ALA -SER ·VAL· GL Y-ASP-ARG-VAL -THR-
`
`40
`30
`ILE-THR-� ARG-ALA-SER-GLN-SER-ILE-ASN-THR-TRP-LEU-ALA-TRP·TYR-GLN-GLN-LYS-PR0-
`60
`50
`GLY-LYS-ALA-PRO-LYS-LEU-LEU-MET-TYR-LYS-ALA-SER-SER-LEU-GLU·SER·GLY-VAL-PRO·SER·
`
`80
`70
`ARG-PHE-ILE-GLY-SER-GLY-SER-GLY-THR-GLU-PHE-THR-LEU-THR-ILE-SER-SER-LEU-GLN-PR0-
`
`90
`100
`ASP·ASP-PHE·ALA-THR-TYR·TYR-�-GLN-GLN-TYR-ASN-SER-ASP-SER-LYS-MET-PHE-GLY-GLN-
`110
`120
`GLY·THR·LYS-VAL-GLU-VAL-LYS·GLY·THR-VAL·ALA-ALA-PRO-SER-VAL-PHE-ILE-PHE-PRO-PRO-
`
`130
`140
`SER-ASP-GLU-GLN-LEU-LYS-SER-GLY-THR-ALA-SER-VAL-VAL-�-LEU-LEU-ASN-ASN-PHE-TYR-
`150
`160
`PRO-ARG-GLU·ALA-LYS-VAL-GLN-TRP·LYS-VAL·ASP-ASN-ALA-LEU-GLN-SER-GLY-ASN-SER·GLN·
`
`170
`180
`GLU-SER-VAL·THR-GLU·GLN-ASP-SER·LYS·ASP-SER-THR-TYR-SER-LEU-SER-SER·THR-LEU·THR·
`
`190
`200
`LEU·SER·LYS-ALA·ASP·TYR·GLU-LYS-HIS-LYS-VAL-TYR-ALA-� GLU-VAL-THR-HIS-GLN-GLY·
`210
`214
`LEU-SER· SER-PRO-VAL-THR-L YS-SER -PHE-ASN-ARG· GLY· GLU-�
`
`FIG. 2.-Complete amino acid sequence of the Eu light cha.in. Ha.lf-cystinyl residues a.re in
`boxes and methionyl residues a.re underlined.
`
`portion of the molecule is attached at Asx residue 297.14 In a previous study8 we
`have suggested that glut.amyl residue 356 and methionyl residue 358 may be
`associated with Gm 1 specificities. The sequence of Eu (Gm 4+) between
`residues 211-252 can be compared with the partial sequence of immunoglobulin
`Daw17 (Gm 4-). The presence of arginine in position 214 of Eu and lysine in a
`comparable position of Daw may be ru380ciated with their Gm 4 specificities.18
`Of particular significance is the determination of the point at which Va ends
`and Ca begins. A CNBr fragment comparable to fragment H4 was isolated
`from myeloma protein He which has the same Gm specificity as protein Eu. The
`sequence of the amino terminal portion of the CNBr fragment from He differed
`from that of the H. fragment from Eu. 21
`
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`VOL. 63, 1969
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`81
`
`1
`10
`20
`PCA·VAL·GLN·LEU·VAL·GLN·SER·GLY·ALA·GLU·VAL·LYS·LYS·PRO·GLY·SER·SER·VAL·LYS·VAL·
`30
`40
`SER-�· L YS·ALA· SER· GLY • GLY • THR · PHE ·SER ·ARC· SER ·ALA· I LE· I LE· TRP-VAL • ARG· GLN. ALA·
`60
`50
`PRO· GL Y · GLl'.J • GLY· LEU· GLU · TRP· �-GLY· GLY ·I LE·VAL ·PRO-MET ·PHE ·CLY· PRO· PRO· ASN · TYR ·
`70
`80
`ALA·GLN·LYS·PHE·GLN-GLY·ARG·VAL·THR· ILE·THR·ALA·ASP·GLU·SER·THR·ASN·THR·ALA-TYR·
`90
`100
`�1ET • GLU·LEU· SER· SER ·LEU· ARG ·SER· GLU·ASP· THR ·ALA· PHE · TYR · PHE ·§ ALA· GLY· GLY • TYR •
`110
`120
`CLY· ILE·TYR·SCR·PRO·GLU·GLU·TYR·ASN·GLY·GLY·LEU·VAL·THR·VAL·SER-SER·ALA·SER·THR·
`130
`140
`LYS·GLY·PRO·SER-VAL-PHE·PRO·LEU·ALA·PRO·SER·SER·LYS-SER-THR-SER·GLY·GLY·THR-ALA·
`150
`160
`ALA- LEU· GLY ·§ LEU· VAL· L VS ·ASP· TYR ·PH£ -PRO· GLU· PRO·VAL • THR ·VAL· SER-TRP-ASN· SER·
`170
`130
`GLY·ALA·LEU·THR·SER·GLY·VAL·HIS-THR-PHE-PRO·ALA·VAL-LEU·GLN·SER·SER·GLY-LEU-TYR·
`
`190
`�
`SER· LEU· SER· SER· VAL· VAL· THR ·VAL· PRO· SER· SER· SER -LEU· GLY-THR · GLN • THR · TYR • I LE·�
`210
`�
`ASN ·VAL ·ASN ·HIS· LYS ·PRO· SER -ASN · THR· LYS·VAL • ASP·LYS ·ARG ·VAL· GLU· PRO-LYS· SER-�
`230
`240
`ASP-LYS · THR · ltl S • TltR -�·PRO· PRO-�· PRO· ALA· PRO· GLU· LEU· LEU·GLY· GLY ·PRO· SER ·VAL·
`250
`260
`PHE ·LEU-PHE ·PRO· PRO· L VS· PRO ·l VS ·ASP· THR ·LEU· MET-I LE -SER -ARG-THR-PRO· GLU·VAL • THR •
`
`2SO
`270
`� VAL·VAL·VAL-ASP-VAL-SER-HIS·GLU·ASP·PRO·GLN·VAL-LYS·PHE·ASN-TRP·TYR-VAL-ASP·
`290
`300
`GLY-VAL-GLN-VAL-HIS-ASN·ALA·LYS-THR·LYS-PRO-ARG-GLU·GLN-GLN-TYR-ASX-SER·THR·TYR·
`
`320
`310
`ARG·VAL·VAL·SER-VAL-LEU·THR-VAL·LEU-HIS-GLN-ASN-TRP-LEU-ASP-GLY-lYS-GLU-TvR-LYS·
`330
`340
`§ Lv5-VAL·SER·ASN·LYS·ALA·LEU-PRO-ALA-PR0-IL£-GLU-LYS·THR-ILE-SER-LYS·ALA-LYS-
`360
`350
`GL Y · GLN ·PRO· ARG • GLU ·PRO· GLN-VAL · TYR -THR -LEU-PRO· PRO-SER -ARG-GLU • GLU· MET-THR -LYS-
`370
`380
`ASN · GLN· VAL· SER-LEU· THR ·(@ LEU· VAL· LYS· GL Y· PHE · TYR -PRO· SER ·ASP· I LE ·ALA-VAL ·CLU·
`400
`390
`TRP-GLU·SER·ASN-ASP-GLY·GLV·PRO·GLV·ASN·TYR·LYS·THR·THR-PRO·PRO·VAL-LEU·ASP·SER-
`420
`410
`ASP·GLY·SER·PHE·PHE·LEU-TYR·SER·LYS·LEU·THR-VAL·ASP-LYS-SER-ARG-TRP·CLN-GLU·GLY-
`440
`430
`ASN ·VAL· PHE ·SER-�-SER -VAL· MET· HIS· GLU· ALA· LEV· HI S-ASN-H I S-TYR-THR· GLN-L vs-SER·
`446
`LEV· SER· LEU-SER -PRO· GLY
`
`Ft0. 3.-Complcte amino acid sequence of the Eu heavy chain. Half-cystinyl residues are in
`boxes and methionyl residues are underlined.
`
`A comparison of the sequence of the two fragments from residues 101 to 121
`is given in Figure 4. The sequences become identical at residue 115 (Eu num­
`bering). Further studies21 confirmed that from residue 115 to residue 252 the
`sequence of the He fragment was identical to that of Eu H.. In addition,
`tryptic fingerprints of the Fe fragments from Eu and He were identical. These
`
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`UIOCHEMIS'l'llY: EDEJ,MAN ET Af,.
`
`PROC. N. A. s.
`
`EL
`
`120
`115
`110
`105
`101
`·GLY·ILE·TYR·SER-PRO-GLU·GLU·TYR-ASN-GLY·CLY·LEU·VAL·THR·VAL·SER·SER·ALA·SER·THR·LYS·
`
`H(
`
`· THR ·LEU -ALA· PHE ·ASX -VAL· TRP· Gl Y · GLX · Gl Y · THR -LYS· VAL· ALA· VAL· SCR ·SER ·ALA· SER· THR· l YS·
`
`F1<;. 4.-Cornparison of the am ino acid sequence of !he En heavy chain from re:<i<lue 101-121
`with the corre.�ponding seq11e11ce
`
`of the heavy rhain of myeloma protein He.
`
`data suggest that the transition between Vu and C11 is located in the vicinity of
`residue 114 (Eu numbering). Studies on a number of additional proteins and a
`search for Vn region subgroups7 will be required to locate this point definitively.
`Discussion.-The present studies provide proof of the covalent structure and
`arrangement of chains in -yGl immunoglobulin. The half-molecule of Eu is the
`largest protein unit (446 + 214 residues) for which a complete amino acid
`sequence has been determined. In a protein of this size, one cannot neglect the
`possibility of small errors in sequence assignment; for this reason we arc carrying
`out a number of further checks using varioui:; methodi:; of peptide cleavage and
`fractionation.
`One of the most striking features of the immuuoglobulin molecule that emerges
`from the completed sequence is the sharp demarcation of its polypeptide chains
`into linearly connected regions that are associated with different. functions.
`Variations in the sequences of paired VH and V L regions for the function of
`antigen binding in the selective immune response, and at the same time, conser­
`vation of sequence in Cn and CL regions for other immunological functions appear
`to require special genetic and evolutionary mechanisms. 22
`The amino acid sequence of Eu provides convincing evidence that. the immuno­
`globulin molecule evolved by successive duplication of precursor genes. 23• 24 Our
`analysis of a complete heavy chain ha:s revealed an :idditional homology region
`(CHl), the structure of which was not previously known. Earlier comparisons7• 8
`of polynucleotide sequences corresponding to both chains of Eu showed evidence
`of homology between Vu and V L and homologies among Cr., Cn2, and CH3. A
`complete comparison of the amino acid sequences of CL, CHl, Cn2, and Cu3 is
`In a stretch of 100 residues, any two regions are identical in
`given in Figure 5.
`29 to 34 positions. It is noteworthy that the stretch in the heavy chain from
`residue 221 to 233 which contains the interchain disulfide bonds; hai:; no homol­
`ogous counterpart in other portions of heavy or light chains.
`In the data accumulated so far, little or no homology has been found between
`V and C regions. This prompts the speculation that V genes and C genes
`diverged early in the evolution of antibodies to serve two major groups of func­
`tions: antigen recognition functions (ARF) and effector functions (BF) such as
`interaction with cells and complement. The order of emergence of Cr, genes or
`CH genes from a precursor gene is not apparent from the data. A comparison
`with sequences ofµ, a, o, or E chains may indicate similarities in their Vu regions25
`and may show whether any of the homology regioni:; are conserved in constant
`regions of heavy chains of these classes. 26
`Early evolutionary divergence of V and C genes i:-; consistent with the evi­
`dence27 · 28 that each chain is specified by two genes, V and C, and the hypothe­
`sis7· 22· 29 that V gene episomcs nrc translocated to C genes to form a single VC
`
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`VoL. Ga, 1969
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`83
`
`<RESIDUES 109-214>
`
`EU C
`L
`EU C
`l <RESIDUES 119-220)
`H
`2 <RESIDUES 234-341)
`EU C
`H
`3 tRESIDUES 342-44bl
`EU C
`H
`
`110
`THR VAL ALA ALA PRO SElt
`SER THR LYS GLY PRO SER VAL PHE PRO LEU ALA PRO
`
`120
`I LE PHE PRO PRO
`
`LEU LEU GL Y GL Y PRO SER VAL PHE LEU PHE PRO PRO LVS
`
`GLH PRO ARG GLU PRO GLH VAL TYR THR LEU PRO PRO SER
`
`130
`LEU LYS SER GLY THR ALA SER VAL VAL CVS LEU LEU ASN ASH PHE
`
`ASP GLU GLN -
`SER LYS SER -
`THR SER GLY GLV THR AL.\ ALA LEU CLY CVS LEU
`PRO LYS ASP THR LEU MET I LE SER ARG THR PRO GLU VAL THR CVS
`ARG GLU GLU -
`MET THR LVS ASH GLH VAL SER LEU THR CVS
`
`LYS ASP TYR
`
`VAL ASP VAL
`
`LYS GLY PHE
`
`PRO ARG GLU ALA LYS VAL -
`PRO GLU PRO VAL THR VAL -
`
`150
`GLN TRP LYS VAL .\SP ASN A
`
`GLN SE
`
`SER TRP ASN SER - GLV ALA LEU THR SER GL V
`
`HIS GLU ASP PRO GLN VAL LYS PHE ASN TRP TYR \l.\L ASP GLV - VAL GLN VAL HIS
`PRO SER ASP I LE ALA VAL -
`
`GLU TRP GLU SER ASN ASP -
`
`GL Y GLU PRO GLU
`
`170
`lbO
`SER GLN GLU SER VAL THR GLU CLN ASP SER LVS ASP SER THR TYR SER LEU SER SER
`- VAL HIS THR PHE PRO ALA VAL LEU GLN SER - SER GLV LEU TYR SER LEU SER SER
`SN ALA L VS THR L YS PRO ARC CLU CLN GLN TYR - ASP SER THR TYR ARC VAL VAL SER
`S1j TYR LVS THR THR PRO PRO VAL LEU ASP SER -
`SP CLY SER PHE PHE LEU TYR SER
`
`190
`180
`THR LEU THR LEU SER LYS ALA ASP TYR CLU LYS HIS L VS VAL TYR ALA CVS CLU VAL THR
`\'AL VAL THR \/AL PRO SER SER SER LEU CLY THR CLN - THR TYR ILE CVS ASN VAL ASN
`l\L LEU THR V.\L LEU HIS CLN ASN TRP LEU ASP CLV L VS CLU TYR L VS CVS LYS VAL SER
`LYS LEU THR VAL ASP LVS SER ARC TRP CLN CLU GLV ASN VAL PHE SER CVS SER \/AL MET
`
`200
`CLN CLY LEU SER SER PRO VAL 'THR - LYS SER PHE -
`LYS PRO SER ASN THR LYS VAL -
`ASP L VS ARG VAL -
`CLU PRO LYS SER CVS
`ASN LYS ALA LEU PRO ALA PRO I LE - GLU L VS THR I LE SER LVS ALA LYS GL �
`GLU ALA LEU HIS ASN HIS TYR THR CLN l VS SER LEU SER LEU SER PRO C.C V
`
`210
`ASN ARC CLV GLU CVS
`
`-
`
`FJG. :3.-Comparisoo of t.he amino acid sequence of CL, C:�l, CH2, and CH3. Deletfons
`indi­
`cat.ed by dashes have been inl.roduced to maximize the homology. Identical residues are darkly
`shaded; both dark and light. shading are nsed lo indicate identitie:< which occur in pairs in the
`Rame positions.
`
`gene in lymphoid cell precursors. Dreyer and Bennetta.> have previously sug­
`
`
`
`of C genes and, more recently,
`this has been abandoned in
`gested a translocation
`
`31 Translocation of genes may be the
`
`
`favor of a detailed "copy-splice" mechanism.
`
`
`
`
`basis of the phenomena of clonal expression and allelic exclusion in antibody
`
`
`Irreversible differentiation and commitment of a lymphoid pre­
`production.
`cursor cell may thus occur at the time of gene translocation.
`
`
`The alignment of disulfide bonds, the arrangement of symmetry axes, and the
`
`fact that proteolytic enzymes cleave the molecule to produce Fab, Fe and Fe'
`
`fragments32 suggest that each homology region may be folded in a compact
`
`
`domain29 stabilized by a single intrachain disulfide bond and linked to neighbor­
`
`
`ing regions by less tightly folded stretches of polypeptide chain. Such domains
`
`
`
`would have similar but not identical tertiary structures, and each domain would
`
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`84
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`
`PROC. N. A. 8.
`
`contribute to at least one active site mediating a function of that class of im­
`munoglobulin. This domain hypothesis is consistent with the hypothesis that
`the molecule evolved by gene duplication as well as with the translocation
`hypothesis. As mentioned above, comparison of the structure and function of
`CH regions in different immunoglobulin classes should reveal whether addition or
`deletion of homology regions and corresponding domains is a major mechanism
`in the evolution of these classes.
`Support for the domain hypothesis would come from finding that limited
`proteolysis of Fab fragments yields fragments containing halves of the Fd frag­
`ments. Similar treatment of Bence-Jones proteins may produce V L and CL
`fragments.33 Additional evidence may come from location in Cu of sites for
`complement fixation and skin fixation. Final proof or disproof of this hypothesis
`obviously rests on the results of X-ray crystallographic analysis.
`It is clear
`that a rotation axis passes through the disulfide bonds linking the heavy chains
`and there may be an axis of pseudosymmetry between the light and heavy chains.
`The locations of the interchain and intrachain disulfide bonds, the extensive
`homologies, and the alignment of the light and heavy chains with each other
`suggest that the overall relationships described above will be conserved in the
`three-dimensional structure regardless of the details of folding.
`The exact contribution of the variable regions to the antigen combining site
`must also await analysis of the three-dimensional structure.
`It is known that the
`Fab fragment contains both V L and V H regions, and affinity-labeling experi­
`ments21 indicate that tyrosyl residues34• 3� in these regions are directly involved
`in antigen binding. The constancy of the disulfide bonds in VH and V L regions
`and their coordinate location suggest the possibility that the site is fixed by these
`bonds and that variations in the branches of a chain connected by each bond
`may be sterically arranged around the bond as a center. The closely homologous
`CL and Cttl regions may serve additionally to stabilize the structure in the face of
`the variation, so that both V Land V H can participate in the site.
`In contrast to the diversity of V regions, the origin of which is still unknown, 22
`the C regions appear to be quite stable in various animal species. Recent
`studies8 show striking resemblances in the Fe portions of rabbit23 and human
`'YG immunoglobulin.
`In this respect, C regions, like enzymes, may have evolved
`to interact with specific molecules, e.g., those of the complement system. The
`presence of genetic differences in C regions has not so far been related to their
`function, but there is no reason to expect that the origin of variations in C regions
`will differ from other genetic polymorphisms.
`
`We are deeply indebted to Dr. Jack Brook for supplying Eu plasma. We are grateful
`to Miss Joan Low, Miss Catherine Volin, and Mrs. Hclvi Hjelt for their expert technical
`assistance.
`
`•Supported by grant GB 6546 from the National ScienC'c Foundation and by grant AM
`04256 from the N a.tional Institutes of Health.
`1 For a general review, see Cold Spring Harbor Symposia on Quantitative Biology, vol. 32
`(1967), and Nobel Symposium S: Gamma Globulins, St.ruclure and Control of Biosynthesis, ed.
`J. Killander (Stockholm: Almqvistand Wiksell, 1967).
`z Edelman, G. M., W. E. Call, M. J. Waxdal, and W. H. Konigsberg, Biochemistry, 7, 19!i0
`(1968).
`
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`85
`
`a Waxdal, M. J., W. H. Koniy;;berg, W. L. Henley, anti G. M. Edelman, Biochemistr-y, 7, l!J.iH
`(1968).
`• Waxdal, M. J., W. H. Koni�berg, 1111rl G. M. Edelman, Biochemistry, 7, 1967 (1!�68).
`•Gall, W. E., B. A. Cunningh11.m, l\l. J. Waxd11.l, W. H. Konig>;berg, and U. M. Edelman,
`Biochemistry, 7, 1973 (1968).
`6 Cunningham, B. A., P. D. Gottlieb, W. H. Konigi;berg, and G. M. Edelman, Biochemistry,
`7, 1983 (1968).
`7 Gottlieb, P. D., B. A. Cunningham, M. J. Waxdal, W. H. Konigsberg, and G. M. Edelman,
`61, 168 (1968).
`these PROCEEDINGS,
`a Rutishauser, U., B. A. Cunningham, C. Bennett, W. H. Konigsberg, and G. M. Edelman,
`
`these PROCEEDINGS, 611 1414 (1969).
`9 Bennett, C., W. H. Konigsberg, and G. M. Edelman, manuscript in preparation.
`10 Gall, W. E., and G. M. Edelman, manuscript in preparation.
`11 Brown, J. R., and B. S. Hartley, Biochem. J., 101, 214 (1966).
`11 Waxdal, M. J., W. H. Konigsberg, and G. M. Edelman, in Cold Spring Harbor Symposia tm
`Quantitative Biology, vol. 32, 53 (1967).
`u Pink, J. R. L., and C. Milstein, Nature, 216, 941 (1967).
`14 Inasmuch as the mechanism of attachment of the carbohydrate is unknown, we have not
`specified whether residue 297 is Asp or Asn. This residue begins the tripeptide sequence Asx­
`Ser-Thr (residues 297-299), which is the same as the Asx-X-Thr sequence found at the point
`of attachment of the carbohydrate portion of several other glycoproteins. 16• 16
`16 Eylar, E. H., J. Theoret. Biol., 10, 89 (1966).
`14 Catley, B. J., S. Moore, and W. H. Stein, J. Biol. Chem., 244, 933 (1969).
`11 Steiner, L. A., and R.R. Porter, Biochemistry, 6, 3957 (1967).
`1• The association with Gm 1 specificities is based on definitive studies. 11• •0 Additional
`evidence is required for the correlation of sequence variation with Gm 4 specificities.
`11 Thorpe, N. 0., and H.F. Deutsch, Immunochemistry, 3, 329 (1966).
`20 Frangione, B., C. Milstein, and J. R. L. Pink, Nature, 221, 145 (1969).
`ii Cunningham, B. A., M. Pflumm, U. Rutishauser, and G. M. Edelman, manuscript in
`preparation.
`u Edelman, G. M., a.ndJ. A. Gally, in Brookhaven Symposia in Biology, No. 21, 328 (1968).
`u Hill, R. L., R. Delaney, R. E. Fellows, Jr., and H. E. Lebovitz, these PROCEEDINGS,
`56,
`1762 (1966).
`u Singer, S. J., and R. F. Doolittle, Science, 153, 13 (1966).
`16 Wikler, M., H. Kohler, T. Shinoda, and F. W. Putnam, Science, 163, 75 (1969).
`ie For generality, we have used CL and Cal, C82, etc., to designate homology regions. If
`such regions are found in other classes, a more specific nomenclature, e.g. C,., C-yl, C,,2, etc., and
`perhaps C,.1, etc., may be required. The same obviously holds for V regions.
`27 Hood, L., and D. Ein, Nature, 220, 764 (1968).
`28 Milstein, C., C. P. Milstein, and A. Feinstein, Nature, 221, 151 (1969).
`29 Edelman, G. M., and W. E. Gall, Ann. Rev. Biochem., 38, (1969), in press.
`ao Dreyer, W. J., andJ. C. Bennett, these PROCEEDINGS,
`54, 864 (1965).
`11 Dreyer, W. J., W.R. Gray, and L. Hood, in Cold Spring Harbor Symposia tm Quantitative
`Biology, vol. 32, p. 353 (1967).
`u Turner, M. W., and H. Bennich, Biochem. J., 107, 171 (1968).
`u Berggll.rd, I., and P. Peterson, in Nobel Symposium S: Gamma GWlru.lins, Structure and
`Control of Biosynthesis, ed. J. Killander (Stockholm: Almqvist and Wiksell 1967), p. 71.
`
`u Singer, S. J., and N. 0. Thorpe, these PROCEEDINGS, 60, 1371 (1968).
`u Although there are no direct data on affinity labeling of human antibodies, by analogy (see
`ref. 24), we would suggest that tyrosyl residues 86 of the light chain and 94 of the heavy chain
`may have a function in the active site.
`
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`BI Exhibit 1091
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