`Vol. 71, No. 3, pp. 845-848, March 1974
`
`Variable Region Sequences of Five Human lmmunoglobUlin Heavy Chains
`of the V Hiil Subgroup: Definitive Identification of Four Heavy
`Chain Hypervariable Regions
`(oiyeloma proteins/amino acid sequences/antibody combining site)
`.
`
`J. DONALD CAPRA AND J. MICHAEL KEHOE
`
`Department of Microbiology, Mount Sinai School of Medicine of the City University of New York, 10 East 102 Street, New York, N.Y. 10029
`
`kappa), and Tur (IgAl kappa) were isolated from serum or
`plasma by zone electrophoresis on polyvinyl copolymer
`("Pevicon") (8). After further purification by gel filtration
`chromatography, they were reduced with 0.1 M P-mercapto(cid:173)
`ethanol and alkylated with iodoacetamide. The heavy
`and light chains were separated by gel filtration in propionic
`acid (9, 10).
`
`Fragment Preparatwn: Heavy chains were treated with
`cyanogen bromide (11) and the resuiting individual frag(cid:173)
`ments purified by gel filtration chromatography in 5 M guani(cid:173)
`dine · HCl. Three proteins (Tei, Zap, and Tur) yielded a
`large N-terminal fragment comprising residues 1-85. Pro(cid:173)
`teins Was and Jon, which contain a methionine residue at
`position 34, gave fragments comprising residues 1-34 and
`35-85. Since alf human JgG myelomas have a methionine at
`position 252, Tei and Was yielded a large fragment comprisi~g
`-residues 86-252. In protein Jon, however, an additional
`methionine was present at position 111. Consequently two
`distinct fragments comprising residues 86-111 and 112-252
`were obtained from this protein. IgAl proteins contain a
`methionine at residue 426 (12) so proteins Zap and Tur both
`yielded a very large fragment composed of residues 86-426.
`
`Sequencing Procedure. Positwns 1-85: On both the intact
`heavy chain as well as on the 1-85 fragment, proteins Tei,
`Zap, and Tur were sequenced 60 steps on the automated se(cid:173)
`quencer (13, 14). Tryptic peptides were prepared and sepa(cid:173)
`rated on Dowex 50 X 4 with a pyridine-formate buffer system.
`In proteins Tei and Zap two invariant peptides were aligned
`by homology alone (70-74 and 75-78) while in protein Tur,
`
`8
`1234~6 7
`I !
`t-t .H
`H I H H
`
`10
`
`,...
`
`Communicated l>y Henry G. Kuniel, ·N01Jem"ber 5, 1973
`
`The variable regions of five human imoiu(cid:173)
`ABSTRACT
`noglobulin heavy chains of the V Hiii subgroup have been
`totally sequenced. Three of the heavy chains belonged to
`the lgG class and two to the IgA class. Examination of
`·these sequences, and comparison with additiona] pllb(cid:173)
`lished heavy chain sequences, showed that a total of four
`hype:rvariable regions is characteristic of human heavy
`ch~in variable regions.
`The relatively con~:rved character of large segments of
`the heavy chain va.riable region was very evident in these
`studies. The conserved segments, which are those sections
`located outside the hypervariable regions, comprise ap(cid:173)
`proximately 65% of the total heavy chain variable region.
`The following general struciural pattern for antibody
`molecules emerges from this and related stud.ies: an over(cid:173)
`all combining region superstructure is provided by the
`more conserved segments while the refinements of the
`active site specificity are a function of hypervariable re(cid:173)
`gions.
`
`The antibody combining site is now believed to reside ex(cid:173)
`clusively in the variable regions of the heavy and light poly(cid:173)
`peptide chains of the immunoglobulin molecule. Evidence
`is accumulating from several laboratories which indicates
`that hypervariable regi<ms within the variable region are
`directly involved in the antibody combining site as well as
`being responsible, at least in part, for the idiotypic determi(cid:173)
`nants of myeloma proteins and specific antibodies (1-5).
`The existence of three hypervariable regions in the vari(cid:173)
`able region of human immunoglobulin heavy chains has been
`established by previous studies from this laboratory. Resi(cid:173)
`dues 31-37 were described as the first hypervariable region
`of the heavy chain (6), and, after fragmentation of IgG
`heavy chains with cyanogen bromide, two additional hyper(cid:173)
`variable regions were localized between residues 86-91 and
`101-110 (7).
`We have now completed the amino acid sequence from
`residues 41 to 84 of the three VHIII proteins originally re(cid:173)
`ported (6, 7) as well as the complete V region sequence of
`two IgA proteins with VHIII variable regions. The data
`make apparent an additional area of sequence hypervari(cid:173)
`ability between residues 51 and 68, thus supporting the ob(cid:173)
`servations of Cebra et al. made on pooled guinea-pig immuno(cid:173)
`globulins (5). When these data on VHIII proteins are in(cid:173)
`cluded with that available for proteins of the VHI and VHII
`subgroups and analyzed by the method of Wu and Kabat (3),
`four distinct areas of sequence hypervariability are observed.
`
`MATERIALS AND METHODS
`
`Myelmna Proteins. Tei (IgGl kappa, Gm az), Was (IgGl
`kappa, Gm az), Jon (IgG3 lambda, Gm g), Zap (IgAl
`
`845
`
`o
`
`~ ro ~ ~ ~ ~ ro ~
`TUBE NUMBER
`F10. 1. Representative ion exchange chromatogram of tryptic
`hydrolysate of the amino terminal (1-85) cyanogen bromide
`fragment of · a chain from protein Tur. Peptides were isolated
`from a Dowex 50X4 column and characterized and analyzed as
`described in the text.
`
`6 .0
`
`"'
`~ 5.0
`t;
`
`I
`
`•
`
`•
`
`rtAJ~~) ~wt~
`
`1 of 4
`
`Celltrion, Inc., Exhibit 1092
`
`
`
`846
`
`Immilnology: Capra and Kehoe
`
`Proc. Nat . .Acad. Sci. USA 71 (1974)
`
`10
`
`20
`
`Zap
`
`'l'ur
`
`Tei
`
`JO
`.
`GLY PRB 'l'BR PRB SER TRR SD ALA VAL TYJt (
`
`40
`50
`J TRP VAL ARG GLlf ALA PRO GLY LYS GLY LEU GLU TRP VAL
`
`- - - SD - - - - - ASP _
`MBT _
`(
`__________ __,ALA TRP MET LYS (
`
`'l'BR SER AJIG PRE
`
`ARG VAL LEU SER SER (
`
`60
`70
`GLY TRP ARG TYJt GLU GLY SBR SER LEU 'l'BR BIS ~ ALA VAL SD VAL GI.If GLY ARG PRE 'l'BR ILE SER MC A.Sii
`GLlf GL11 ALA _ . Asif SER
`PRE
`
`ALA
`
`LYS
`
`ASP 'l'BR
`
`Jon
`
`Zap
`
`TUr
`
`Tei
`
`Wae
`
`Jon
`
`VAL - - - VAL _
`
`GI.If VAL VAL Gw LYS ALA PBB _
`
`ASll - - - AD - - - - - - - - - - - - - -
`
`Zap GU1 PBB
`
`Tur
`
`SER GLY
`
`VAL GUI - - - ALA ILE SD - - - - - ASP - - - - - ALA - - - - - - - - - - - - -
`LEU ASlf ALA - - - ASlf LEU
`PHB _____ ALA - - - - - - - - - - - - - - - - -
`
`Tei
`
`ao
`.
`.
`too
`90
`.
`~~~~~~Tll'RLBU~~uro~uro~~~~'l'BRALAVALTYRTYR~ALA~
`
`Wa• - - - - - - - - - - - - - - - - - -ASB ARG
`Jon
`
`ALA-----------------~
`
`VAL 'l'BR - - - - - - - - - - - - - - - - - - - -
`- - - - - - - - - - - - - - - - - - ILB
`Zap - - - - - - - - - - - - - - - - - - ASN 'l'BR GLY'
`A~ ------------....;...----~
`Tur
`- - - - - - - - - - - - - - - - - - - - - - - GLR A~ - - - - - - - - LEU --------~
`
`110
`120
`~i VAL'l'BR~ALA~ALA~~~~~~VAL~~~~'l'BRLBUVAL'l'BR
`
`WH
`
`PHE ARG GLN PRO PHE VAL GIB (
`
`Jon
`
`_
`
`VAL VAL SER 'DIR [
`
`TBR ARC
`
`Gt.Y GLY 'lYR [
`
`Zap
`Tur
`
`] -
`
`PHB ASP -
`
`PHE - - - - - - - - - - - -
`
`) SER MET ASP - - - - - - - - - - - PRO - - -
`
`- - - ASP - - - - - - - - - - - - - - SER
`
`LEU SER VAL 'DIR _
`
`VAL [
`
`A~ PHE ASP - - - - - - - - - - - LYS
`SER
`FIG. 2. The amino-acid sequence of the variable regions of five human immunoglobulin heavy chains.
`
`the isolation of chymotryptic pept ides established the se(cid:173)
`quence unambiguously. In all cases, tryptic peptides were
`sequenced .in the autOmated sequencer, often using 4-sulfo(cid:173)
`phenylisothiocyanate (Pierce Chemical) on the lysine pep(cid:173)
`tides (15). In proteins Was and Jon, which contained cyano(cid:173)
`gen bromide fragments 1- 34 and 35-85, the first 60 residues
`were established by automated sequencing of the intact.
`heavy chain. Thus, in these two proteins, sequencing
`cyanogen bromide fragment 1- 34 was superfluous since its
`composition agreed with the previously determined sequence.
`
`Fragment 35-85 of proteins Was and Jon was sequenced 35
`and 40 residues respectively; this, together with the C(cid:173)
`terminal tryptic peptides mentioned above gave the com(cid:173)
`plete sequence for this section. Residues 86-1£1: In proteins
`Tei, Was, Zap, and Tur the sequence was established by a
`continuous automated run of 45 steps from residue· 86 into
`the Cal domain. In both Zap and Tur, tryptic digestion and
`isolation of the resulting peptides confirmed a few question(cid:173)
`able positions. In protein Jon, residues 86-111 were obtained
`disulfide linked to residues 1-34 after cyanogen bromide
`
`2 of 4
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`Celltrion, Inc., Exhibit 1092
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`
`
`Proc. Nat. Acad. Sci. UBA 71 (1974)
`
`Human Immunoglobulin Heavy Chains
`
`847
`
`digestion. This sequence was obtained by difference since
`residues 1-34 were known from the initial study of the
`intact heavy chain. Jon fragment 112-253 was subjected to a
`long sequencer run which definitely established the sequence
`of residues 112- 121 as well as providing sequence data into
`the C8 1 domain.
`Ion Exchange Chromatography. An example of a Dowex
`50X4 chromatogram is shown in Fig. 1 for a tryptic digest
`of the Tur 1-85 fragment; 6.5-ml fractions were collected and
`0.1 ml of each fraction analyzed by the fiuorescamine pro(cid:173)
`cedure initially described by Udenfriend et al. (16). Ninhydrin
`analysis was also performed after alkaline digestion of 0.5-ml
`aliquots. In most analyses, only the fiuorescamine procedure
`was employed since it was much more sensitive. As shown in
`Fig. 1, 10 fractions were pooled. Each was subjected to amino(cid:173)
`acid analysis and several useful peptides were isolated and
`sequenced. T-1 (Asn Thr Leu Tyr Leu Gin Hsr) (79-85), T-3
`(Asn Asp Ser Lys) (75-78), T-7 (Gly Leu Gly Trp Val Ser
`Gly Arg) (46-53), and T-10 (Phe Thr Ile Ser Arg) (7CVi4).
`
`RESULTS AND DISCUSSION
`The amino-acid sequences of the variable regions of the
`five human myeloma proteins is displayed in Fig. 2. The
`variability-factor values determined by the method of Wu
`and Kabat (3) for these as well as all the other human V
`region sequences available is shown in Fig. 3. These calcula(cid:173)
`tions were based on 25 sequences from residues 1 to 34, I I
`sequences from residues 35 to 85, and 14 sequences from
`residues 86 to I22. Previous to this study there were only six
`published complete V region sequences, all but one (Nie) of
`the VHI (Eu) or V8 II (Daw, Cor, He, Ou) subgroup (for
`references see legend to Fig. 3). With five additional VHIII
`sequences the variability within and between subgroups can
`now be compared more meaningfully. In addition, with the
`availability of 11 complete sequences and several fragments,
`the Wu-Kabat plot becomes more statistically significant.
`A discussion of the sequences can be conveniently divided
`into those sections of the V region which are relatively con(cid:173)
`stant (1-30, 38-50, 69-83, 92-100, and 11I-I21), and the
`hypervariable regions (3I-37, 51-68, 84-91, and 101-110).
`About 65% of the variable region of the heavy chain shows
`limited variation. In fact, there are 17 positions (14%) which
`have been absolutely invariant in all human heavy chains
`regardless of their V region subgroup assignment. Certain
`positions are subgroup specific since at these positions all,
`or nearly all, of the members of one subgroup have a particular
`amino acid, while members of the other subgroup contain a
`different amino acid. Utilizing the four available VHII
`proteins, positions 3, 9,16, 17, 19, 21, 23, 28, 29, 39, 42, 46, 50,
`80, 81 and 82 appear to be subgroup specific. As noted
`previously, no subgroup specific residues are identifiable in
`the C terminal portion of the V region (7). There are thus
`33 positions (27%) in the V region which are either invariant
`or subgroup specific. A comparison with the published se(cid:173)
`quences of myeloma proteins (I7, 18), pooled immuno(cid:173)
`globulins (5, 19, 20), and specifically purified antibodies (5,
`21-23) from lower species, indicates that the particular
`amino acids found at these positions are characteristic of a
`wide variety of mammals and have been faithfully conserved
`during evolution. Such residues may have extremely im(cid:173)
`portant attributes for variable region function such as, for
`
`GAP
`u
`
`GAP
`IU
`
`GAP
`UIU
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`JO 20 30 40 50 60 70 80 90 IOO 110 120
`POSITION
`Fm. 3. Variability-factor values for the sequences shown in
`Fig. 1 as well as several other published sequences (36) deter(cid:173)
`mined according to the method of Wu and Kabat (3).
`
`example, the provision of a distinct backbone structure which
`is crucial to antibody function.
`As can be seen on inspection of Figs. 2 and 3, about a third
`of the heavy chain variable region can be considered "hyper(cid:173)
`variable." These regions deserve special consideration be(cid:173)
`cause of their specific implications for the formation of the
`antibody combining site, the nature of idiotypic determinants,
`and various theoretical conceptions of the origin of antibody
`diversity.
`In light chains, affinity labels have been localized near or
`within hypervariable regions (23-25), thus providing direct
`support for the general concept that hypervariable regions
`participate directly in the antibody-combining site. For the
`heavy chain, recent work has also been consistent with this
`idea. For example, Ray and Cebra localized affinity labels to
`the first (31-37) and the fourth (101- 110) heavy chain
`hypervariable regions {26), Haimovich et al. (27) localized
`an affinity label to residue 54 of the mouse myeloma protein
`315 (which has anti-dinitrophenol activity), and Press and
`coworkers have localized affinity labels at or near the fourth
`hypervariable region in rabbit antibodies (28). Therefore,
`although the primary structure and affinity labeling studies
`of these proteins was being carried out independently, and
`even in different laboratories in many instances, there is a gen(cid:173)
`eral implication from the experimental observations that the
`same regions of the molecule which show the highest degree
`of sequence variation are near or part of those particular re(cid:173)
`gions of the heavy chain where affinity labels have been local(cid:173)
`ized.
`A second piece of evidence linking the antibody combining
`site to the hypervariable regions has come from comparisons
`of sequences obtained from pooled immunoglobulin heavy
`chains with those of specifically purified antibody heavy
`chains. Sequence analyses of rabbit (29), guinea pig (5), and
`other mammalian heavy chain pools (19), indicate that a
`definitive sequence cannot be obtained within those regions
`which have been identified as hypervariable on the basis
`of studies with myeloma proteins. However, when specifically
`purified antibodies are studied, a single major sequence can be
`determined, as has been shown most definitively by Ce bra and
`his coworkers (5).
`Additional support for the functional significance of hyper(cid:173)
`variable regions has been provided by current notions con(cid:173)
`cerning the tertiary structure of the immunoglobulin mole(cid:173)
`cule. Crystallographic analysis of human immunoglobulins
`has now advanced to the point where it has been possible
`to assign the residues which may line a "pocket" within the
`
`3 of 4
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`Celltrion, Inc., Exhibit 1092
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`
`
`848
`
`Immunology: Capra and Kehoe
`
`Proc. Nat. Acad. Sci. USA 71 (1974)
`
`immunoglobulin molecule which presumably represents the
`combining site itself (30, 31). In ea.ch instance, the major
`residues which line the pocket a.re associable with hyper(cid:173)
`In addition, the conformational models
`variable regions.
`generated by the nearest neighbor calculations of Ka.bat and
`W1i (32) place hypervariable regions in close association
`with the putative combining site.
`There is also growing evidence that at lea.st some of the
`hyperva.riable regions are involved in the idiotypic determi(cid:173)
`nants of myeloma proteins and antibodies. Cross idiotypic
`specificity among the cold agglutinins (33) and the anti(cid:173)
`gamma globulins (34) is believed to be related to the com(cid:173)
`bining site. In at lea.st two distinct anti-gamma globulin
`molecules, the hyperva.ria.ble regions show striking sequence
`similarities (7, 35).
`The genetic origin of hypervariable regions remains un(cid:173)
`clear. The variability within heavy chain hypervariable
`regions seems more marked than that of light chain hyper(cid:173)
`va.riable regions. Of the ll proteins which have now ha.d their
`V regions completely sequenced, if one considers the 43
`hypervariable positions of the heavy cha.in, there are no two
`proteins which have more than 10 residues in common. It
`seems likely that hundreds, or even thousands, of proteins
`would have to be sequenced in order to find two which are
`identical if no preselection bias (such as selection by idiotypic
`antisera or for combining specificity) is involved. This im(cid:173)
`plies either that there are a very large number of germ
`line genes or that somatic processes a.re necessary to explain
`the diversity in the heavy cha.in hypervariable regions.
`Regardless of their origin, the hypervariable regions clearly
`play a. crucial role in the antigen binding function of immuno(cid:173)
`globulin molecules.
`We thank Dr. Henry Kunkel for the subclass and genetic typ(cid:173)
`ing of the myeloma proteins. Bonnie Gerber, Ellen Bogner and
`Donna Atherton rendered invaluable technical assistance. This
`work was aided by grants from the National Science Foundation
`(GB 17046) and the U.S. Public Health Service (Al 09810) and a
`Grantrin-Aid from the New York Heart Association. J .D.C. is the
`recipient of National Institutes of Health Career Development
`Award 6-K4-GM-35, and J.M.K. is an Established Investigator
`of the American Heart Association.
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`Antibody Formation and Structure, Prague.
`3. Wu, T. T. & Kabat, E. A. (1970)J. Exp. Med. 132, 221-250.
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`(1971} Ann. N.Y. Acad. Sci. 190, 371-381.
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`19. Capra, J . D., Wasserman, R. W. & Kehoe, J.M. (1973) J.
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`29. Cebra, J. J., Givol, D. & Porter, R. R. (1968) Biochem. J.
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`32. Kabat, E. A. & Wu, T. T. (1972) Proc. Nat. Acad. Sci. USA
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`33. Williams, R. C., Kunkel, H. G. & Capra, J. D. (1968)
`Science 161, 379-381.
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`(a) Edelman, G. M., Cunningham, B. A., Gall, W. E., Gottr
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`mann, N.(1970) Hoppe-Seyler'sZ. Physiol. Chem. 351, 1591.
`
`4 of 4
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