Scand. }.ImmunoI., Vol. 5, 1976.
`
`Bence Jones Proteins and Light Chains of
`Immunoglobulins
`
`XIV. Conformational Dependency and Molecular Localization of the Kappa
`(x) and Lambda (1) Antigenic Determinants
`
`A. SOLOMON
`
`University of Tennessee Memorial Research Center, University of Tennessee
`Center for the Health Sciences, Knoxville, Tennessee, USA
`
`Solomon, A. Bence Jones Proteins and Light Chains of Immunoglobulins. XIV.
`Conformational Dependency and Molecular Localization of the Kappa (it) and
`Lambda 0.) Antigenic Determinants. Scand. 3. Immrmol. 5. 685-695, 1976.
`
`The region on the light chain molecule responsible for expression of the K and ).
`antigenic determinants was determined by comparative imrnunochemical analyses
`of intact Bence Jones proteins and natLu'al1y occurring or enzymatically derived
`fragments of Bence Jones proteins that lacked extensive portions of the V region
`or part of the C region. The reactivity of these fragments with numerous antisera
`having specificity for light—chain antigenic determinants indicated the essentiality
`of the intact light polypeptide chain for expression of the K and 1 antigenic deter-
`minants. The conformational dependency of the ‘K. and)‘ antigenic determinants
`was also evidenced by denaturation-renaturation studies on X. and J. chains. The
`V domain, C domain, and interdomain ‘switch’ region contribute to the expres-
`sion of x or 1 antigenicity and to certain isotypic and allotypic specificities.
`
`A. Salomon, University of Tennessee Memorial Research Center, Kn0:ttrt7le_. TN
`37920, USA .
`
`A on the basis of the amino acid sequence of
`either the V or C domain (9). However,
`the
`domain or region on the light chain responsible
`for the immunochemical recognition of the it or
`A antigenic determinants has not been estab-
`lished; it is not known whether these determi-
`
`nants reside on the CL or V,‘ or whether their
`expression requires the intact light polypeptide
`chain.
`
`(9,
`Irnmunochemical and structural analyses
`13, 27, 34) of Bence Jones proteins, myeloma
`proteins,
`and Waldenstrom macroglobulins
`have provided evidence that the light p0lypep—
`tide chains of all
`immunoglobulins consist of
`two chemically distinct types designated kappa
`(x) and lambda (A). As evident from sequence
`analyses of homogeneous x and A light chains -
`that is, Bence Jones proteins (12) -— both types
`of light chains share certain characteristic struc-
`tural features: each type has an amino—terminal
`portion of calf)? residues of variant sequence
`(V1) and a carboxyl-terminal portion of N107
`residues of constant sequence (CI). The V]_ and
`CL are under separate genetic control (17) and
`exist as two compact domains (designated V
`and C,
`respectively)
`linked by an extended
`c\o10—residue section of the light polypeptide
`chain termed the ‘switch’
`region (31, 35). A
`given light chain may be distinguished as x or
`
`The relation between immunoglobulin struc-
`ture and antigenicity has been established
`through studies of the naturally occurring or
`enzymaticaily derived fragments and the chem-
`ically
`produced
`subunits
`of
`homogeneous
`immunoglobulins (Bence Jones proteins, mys-
`loma proteins, and Waldenstriim n1acroglobu—
`lins) characteristically found in association
`with multiple myeloma and related plasma cell-
`lymphocyte dyscrasi-as (43). The identification
`Mylan v. Genentech
`and characterization of urinar
`low-moleculif
`Mylan V.
`enentec
`IPR2016-00710
`Genentech Exhibit 2089
`Genentech Exhibit 2089
`
`
`IPR2016-00710
`
`

`
`536 A. Solonwn
`
`weight proteins antigenically related to the V
`or C regions of Bence Jones proteins (2, 4, 7,
`10, 44, 4-8, 52, 53, 56), the availability of un-
`usual Bence Jones proteins with extensive V re-
`gion deletions (IS, 42), and the ability to cleave
`specifically Bence Jones proteins into V]_- and
`CL~related fragments (20, 45) have provided
`the means for determining immunochemically
`the region on the light-chain molecule respon-
`sible for the x and /\ antigenic determinants.
`
`MATERIALS AND METHODS
`
`Proteins. Urine samples containing Bence
`Jones proteins and fragments of Bence Jones
`proteins were obtained from our patients with
`multiple myeloma. The specimens were collec-
`ted without preservative and were maintained
`at 0"C to 4°C throughout the 24-h collection
`period. Subsequently, a sample of each 24-h
`specimen was frozen and stored at -—20°C or
`—70°C. The remaining urine specimen was 1yo-
`philized after extensive dialyses at 4"C in 23!
`32 Visiting tubing (Union Carbide Corp., New
`York) against deionized double-distilled water.
`Preparative procedures. Bence Jones pro-
`teins and fragments of Bence Jones proteins
`were isolated from urine specimens by zone
`electrophoresis on starch or Pevikon blocks and
`then purified by gel filtration through poly-
`acrylamide columns as previously described
`(45). The A. chain Mcg (14), A chain Sm (15),
`and 2: chain Sac (42) were furnished by Dr. Al-
`len B. Edmundson, Dr. Elliott F. Osserrnan.
`and Dr. Dorothy M. Parr,
`respectively. The
`methods used for the preparation and isolation
`of light chains and of the papain-derived Fab
`fragment of F1] yG—globulin were as described
`previously (45).
`The Bence Jones proteins were cleaved by
`pepsin into constant half-related
`and va-
`riant half-related (VI?)
`fragments (45). The
`digestions were performed with an enzyme to
`protein ratio of 1:100 in 0.05M glycine—I-ICl
`buffer, pH 3.4, at 37° C (or at 55” C) and ter-
`minated by raising the pH of each reaction
`mixture to neutrality by adding 1M Tris base.
`These light chain fragments were isolated by
`
`zone electrophoresis and purified by gel filtra-
`tion (45).
`Immmzocbemicai procedures. Antisera were
`prepared in albino New Zealand rabbits to x-
`and )\—type Bence Jones proteins,
`to the light
`chains and the Fab fragment derived from F11
`-yG-globulin, and to intact FII yG~globulin.
`The methods used for the preparation of anti»
`sera and for the immunoelectrophoretic and
`immunodiffusion analyses were as described
`previously (46). The Inv typing of x chains
`was performed by Dr. Arthur G. Steinberg.
`
`RESULTS
`
`Urine specimens from certain of our patients
`with multiple myeloma and Bence Jones pro-
`teinuria who have received high doses of corti-
`costeroids as part of their treatment regimen
`were found to contain a low-molecular-weight
`protein related to the Bence Jones proteins but
`not identical
`to either the VL or the CL (48).
`The new components, designated Cf‘, were
`found to be structurally and antigenicaliy most
`closely related to the carboxyl-terminal half of
`the light polypeptide chain, although each com-
`ponent was 6 to 25 amino acid residues longer
`than the C,‘-related fragment derived in vitro
`by peptic cleavage (CE) of the light chain (48).
`The amino-terminal sequences of CI‘:
`isolated
`from urine specimens of Patients Wins, Oak,
`and Edw and the C E prepared by pepsin cleav-
`
`Table I. In vivo (Cf) and in vitro (Q) constant—half—
`related fragments of X Bence Jones proteins
`Amino-terminus
`
`Protein
`
`Ct Wfms
`S;
`W
`L
`C}: Len
`
`Residue
`
`Position?
`
`Asp
`Tl;1'aflIlE
`Ile
`
`92
`97fl10061"|'
`1
`117
`
`1- The numbers indicate the position of the residues
`along the amino acid sequence of M Bence Jones
`protein Roy (9).
`H‘ Protein Oak consisted of two components of ap-
`proximately equal concentration; the amino-termini of
`components one and two corresponded to positions 97
`and 106, respectively.
`
`

`
`Lacalizanhrt qf it and 1 Ar:rz'gem'c Deterriiirzants
`
`637
`
`Table II. Amino-terminal sequences of ‘K Bence Jones protein fragments
`
`92
`
`I00
`
`VIC
`
`H0
`
`I20
`
`
`
`Cr wms Asp Ile Phe Pro Gly Thr Phe Giy Gin Gly Thr Lys Val Glu lie I.ysArgThrVal Alahla Pro ScrVulPheIlc Phc Pro Pro
`‘l
`CE Oak 1
`Phe Gly
`cg Oak 2
`CE Edw
`
`CE LCD '*"""j
`1" The solid lines indicate sequence identity to CE Wms.
`
`age of Bence Jones protein Len were deter-
`mined by Dr. J. Donald Capra. The amino-
`terminal residues and sequences of the C: com-
`ponents and the CE fragment are given in
`Tables I and II.
`
`The reactivity of the CE components Wms,
`Oak, and Edw and the CE Len fragment was
`compared with that of intact x Bence Jones
`proteins Wms, Oak, Edw, and Len by immu-
`noelectrophoretic analyses of urine specimens
`Wms, Oak, and Edw (containing both Ci‘: and
`Bence Jones protein) and of a sample of Bence
`Jones protein Len cleaved partially by pepsin
`into CE and VE (45). Antisera prepared
`against
`the homologous Bence Jones proteins
`Wms, Oak, Edw. and Len, and antisera prepar-
`ed against 35 heterologous x Bence Jones pro-
`teins were utilized for
`these analyses. With
`anti-homologous protein antisera, the Cl‘: com-
`ponents and the CE fragment were antigeni-
`
`cally cleficient as compared with the intact
`Bcnce Jones protein (Fig.
`1); absorption of
`these antisera with a heterologous x chain left
`reactions only with the homologous
`intact
`Bence Jones proteins. Striking differences in the
`reactivity of the CL—related components were
`found with the .1nti—heterologous x chain anti-
`sera. Certain antisera reacted weakly or not at
`all with the C: components and the CE fr-ag~
`ment. Differences were also evident in the in-
`
`formed by the
`tensity of the precipitin arcs
`different CI’: components. The results obtained
`with the homologous and two representative
`heterologous anti-x chain antisera are also
`shown in Fig. 1. With the anti-3 Isr antiserum,
`the CE components and the CE fragment reac-
`ted with equal intcnsity, each forming a precip-
`itin reaction of identity with the intact Bence
`Jones protein. However, the anti-x Bre antise-
`rum did not react at all with CE Len and reac-
`
`WMS
`
`EDW
`
`LEN
`
`— nnla-hornol BJP
`
`- arm-Klsu
`
`- - onlivltflre
`
`
`
`ll
`
`ll
`
`-
`
`T
`
`T
`
`cf B.JP
`
`cf
`
`am
`
`3.19
`
`cf
`
`c{
`
`3.19 v[
`
`l. Immunoelectrophoretic analyses of intact polypeptide chains and related fragments of four at Bence
`Fig.
`Jones proteins. The antigen wells under the designations WMS, OAK, and ED W’ contained urine specimens
`from Patients Wrns, Oak, and Edw, respectively; the antigen wells under the designation LEN contained a
`sample of Better: Jones protein Len subjected to limited pepsin proteolysis at 37'C. Each sample was tested
`against an antiserum prepared to the homologous Bence Jones protein (anti-Iiomoi. HIP) and against antisera
`prepared to heterologous it Bence Jones proteins Isr and Bre {anti-it Isr and amine Bra). B_?P = Bence Jones
`protein; C: = constant-half-related light-chain component found in vivo; Cf and
`-= constant-hall? and
`variant-half-related light—chain fragments, respectively, produced in vitro by pepsin pruteolytic cleavage of
`Bence Jones protein. The anode was located to the left.
`
`
`
`

`
`688
`
`.4 .
`
`.‘a'o.’ormIn
`
`A’ win:
`
`xJo
`
`Irlsr
`
`xouk
`
`KEdw
`
`K Len
`
`a mi - K Bre
`
`Fig. 2. Imntunodiffusion analysis of six 7. Hence Jones
`proteins. The peripheral antigen wells contained six
`ditT¢-.-rent Illlti-Inc Bence Jones proteins (3-:
`lY’ms, X Oak,
`7. }:'Jtt'. K Len,
`1-: Isr. and K _?'m. The central anti-
`serum well contained an antiserum prepared against
`7. Hence Jones protein lire {un.'.:'-st. Hrs).
`
`ted only wenltly with CE Edw and CE Oak,
`but :1 strong reaction occurred with C: Writs.
`The ‘.1I1tl-x Bre antiserum was él potent Jtiti-:4
`itnti.-serum;
`the prccipitin re-.1ctions ohtailted
`with six x Bence Jones proteins are shown in
`the imniunodiffusion analysis depicted in Fig.
`2. Differences in the reactivity among the CE
`components were evident with many other
`.tnti-x-chitin .1ntiser:1:gener'.1lly, the intensity of
`
`WMS
`
`OAK
`
`precipitin reactions among the C,‘-related frag-
`ments followed the order: (1? Writs 7* C : Oak
`F‘ CI’: Edw >
`Len.
`Other types of ;tntiser-.1 with specificity for
`:4 antigenic Llt.'tI..‘I‘I‘nl1‘l'.11‘lI.‘; also showed this same
`pattern of reactivity with the CL—rel-ated comv
`ponents, as illustrated in Fig. 3 by the 1'L“.1C[l011S
`obtztined with ::Lntiser.t to int-.ict Fl] y(}-glohu-
`lin and its subunits. The anti-FIE light Cl‘l'.1lI'lS,
`‘.'|.l'.'l.Ci-F11 Fab, and anti-Fll y(}-globulin Cl,I‘l[lSCI".l
`did not form visible precipitin reitctions with
`CIT’) Len, and reacted only wealtly with C:
`Edw and C: Oak; n more intense precipitin
`reaction occurred with CE Writs. All
`three
`itntisern reacted strongly with intact x (as well
`.15 intztct A) tihains and, further, had the capac-
`ity to distinguish between ;—_r and .\ light chains.
`The results of
`immunodiffusion attalysis of
`three 9-: and three A Benet’ Jones proteins with
`the anti—Fll Fab antiserum are shown in Fig. 4.
`We lt-ave not -.15 yet detected C[—type com-
`ponents in urine specimens from our patients
`excreting .\ Bence Jones proteins. For this rea-
`son we treated six different A Bence Jones pro-
`teins with pepsin :tt 3? C or at 55f‘C (40) to
`generate
`components in an effort to test the
`reactivity of our anti—;\ chain antisera with CL-
`relatcd fragments. The reactivity of intact A
`
`
`
`‘ Ont: ' homo! BJP
`
`- nnrl-Fll L‘Ch0If|5
`
`- nnll-Fl! Fab
`
`- uni:-FE
`
`cf 3.19
`
`cf
`
`asp
`
`BJP
`
`T
`
`C
`
`BJP
`
`F
`VL
`
`Immunoelectrophoretic analyses of intact polypeptide chains and related fragments of {out it. Hence
`liig. 3.
`Jones proteins. The antigen wells under the designations llt".MS, ()riK. and I.:‘1_)ll" contained urine specimens
`from Patients Wms, Oak. and Etlw. respectix-'el_v‘; the antigen wells under the designation 1.l:‘.'\’ contained a
`sample of Bence Jones protein Len subjected to limited pepsin proteol}.-‘sis at 37 C. Each sample was tested
`against an antiserum prepared to the homologous Hence Jones protein (tm::'-Jmmol. B71") and against antisera
`prepared to the light chains of l~‘II ~,rG-globulin (arzti-F1! I.-chairtt), the papain~derived 1-‘ab fragment of FII
`-{G-globulin (orm'—I*'H Pub), and to FII -{G-globulin (wm'~FU}. B31’
`Be|1ce]ones protein; C; -
`= constant-
`hall-related light-chain component found in vivo; C}: and V‘: : constant-half- and variant-half—related light-
`chain fragments. respectively. produced in vitro by pepsin proteolytic cleavage of Bence Jones protein.
`The anode was located to the left.
`
`
`
`

`
`Localization of st and }. zirztfgertic Dererrrtirtarzts
`
`689
`
`Itwrns
`
`G)
`
`G)
`
`— Dnl:- IMCQ
`
`- unit-FII L-chums
`
`_
`
`_
`
`ant:
`
`It}-‘Ill
`
`Pepsm Diqesl
`[55.CJ"Mc°
`
`a.Hil
`
`(Len
`
`LCIE
`
`anti- FII Fob
`
`Fig. -1. Imrnunodiffusion at'1al_\-‘sis of three K and three
`9. Benee Jones proteins. The peripheral antigen
`wells contained three I Bence Jones proteins (1:
`lY”1us, V. Eda», and 1-: Len) and three 3'. Bence Jones
`proteins (1 Meg, 3. C12, and '2. Hill. The central
`well contained an antiserum prepared against
`the
`papain-derived Fab fragment of FII -{G-globulin
`(mm?-FH Fab}.
`
`Bence Jones protein Meg and C11: Meg (formed
`by partial peptic proteolytic cleavage of pro-
`tein Meg) is presented in Fig. 5. This immuno-
`electrophoretic analysis shows that the antise-
`rum prepared to Bence Jones protein Meg reac-
`
`T
`T
`sup
`C:
`intact
`lmmunoelectrophoretic analysis of
`5.
`Fig.
`polypeptide chain and constant-half~related fragment
`of a 1 Bence Jones protein. The antigen wells contain-
`ed a sample of ‘It Benee Jones protein Meg subjected
`to Iimited pepsin proteolysis at 55'C. The upper,
`middle, and lower antiserum troughs contained anti-
`sera prepared against
`it Hence Jones protein Meg
`(cmu'—). Meg), It Renee Jones protein Iilil (tn-m"-}t H:'!')_,
`and the light chains isolated from FII -[G-globulin
`(tmn'-FH I.-clm:'m_1, respectively.
`
`ted only with the intact protein and not with
`the CIT: component. The antiserum prepared to
`A Bence Jones protein I-lil recognized both in-
`tact protein Meg and its CE, and, although not
`evident in the figure, the C E component form-
`ed a precipitin reaction of nonidentity with
`the intact Benet: Jones protein. None of 20 an-
`tisera prepared against other A Bence Jones
`proteins formed a visible precipitin reaction
`
`Immunodiffusion
`6.
`Fig.
`analysis of
`intact polypep-
`tide ehains and constant-
`half-related
`fragments
`of
`three A Benee Jones proteins.
`The peripheral antigen wells
`in both patterns contained
`intact ‘A. Benee Jones proteins
`(1 LET), 1 Meg, and 1 Chi}
`and their 55’C pepsin-de-
`rived
`constant-half-related
`
`fragments (Cf Lev, CE Meg.
`and CE C19). The central well
`in the pattern on the left con-
`tained an antiserum prepared
`against It Bence Jones pro-
`tein Hil
`(‘§nt:'—). Hit). The
`central well
`in the pattern
`on the right contained the
`same
`antiserum absorbed
`with intact 1 Benee Jones
`proteinLev(anrt'-3t.Hi!E).
`
`
`
`cp Cl
`'-
`
`e
`
`.
`.
`0711'‘ NH”
`
`"
`.
`.
`anlri ‘H’! A
`
`
`
`

`
`690
`
`A . Solomon
`
`WMS
`
`ll
`
`C
`
`ii
`
`— on1i—u.Jo A
`
`- anti-homol. BJP
`
`- Ol'|il"KJO
`
`1
`
`i
`
`"
`
`t
`
`I
`
`cf sap
`
`it
`
`our
`
`34?
`
`cf
`
`c[ we v[
`
`Fig. 7. Immunoelectrophoretic analyses of intact polypeptide chains and related fragments of four 2. Hence
`Iones proteins. The antigen wells under the designations l-l5"M.S'_. OAK, and EDW contained urine specimens
`from Patients Wt-ns, Oak. and Edw, respectively; the antigen wells under the designation LE.’\." contained
`a sample of Hence Jones protein Len subjected to limited pepsin proteolysis at 37"C. Each sample was tested
`against an antiserum prepared to the homologous Bence Jones protein (am!-home}. B_?'P) and against an anti-
`serum prepared to 2. Hence Jones protein Jo (arari-it 3'0), and the anti-it Jo antiserum absorbed with a heterolo-
`gous it Bence Jones protein (amt-x_'7o ii). B31’
`-_- Renee Jones protein;
`= constant-half-related light-chain
`component found in vivo; (if and FE
`. constant-half— and variant-hall-related light—chain components,
`respectively, produced in vitro by pepsin proteolytic cleavage of Bence ]ones protein. The anode was to the
`left.
`
`formed from 5
`Meg or with the C)’.
`with
`other A Bence Jones proteins. This lack of reac-
`
`tion was not related to the CA isotype (43). As
`in the case of protein Meg.
`the CE derived
`from other A chains was not
`recognized by
`antisera to the homologous Bence Jones pro-
`teins or by the anti-Fll
`light chains, anti-FII
`
`I-7-ah, or anti-Iill yG-globulin antisera. Usually,
`It chains are more resistant
`than 2 chains to
`
`cleavage by endopeptidztses; specific proteoIy-
`sis o1c:\ chains into VI_ and CL is Facilitated by
`prior reduction and alkylation of the proteins
`(20. 45). Pepsin cleaves :4 chains between posi-
`tions 116 and I1? in the C dorn-‘tin (38). Al-
`though the specific site of .\-chain clealvitgc by
`pepsin is still to be established, our own studies
`and other data (21) indicate that the sequences
`of C)’ fragments derived from A Benee Jones
`proteins include the entire C region and also
`extend \~4 to 10 residues into the V region.
`The anti—i\ Benee jones protein Hil antiserum
`was unique in its capability to react with C].-
`related Fr-agnients formed by pepsin cleavage of
`A chains. A comparative iinmunodiffusion anal-
`ysis of three A Bence _]ones proteins and their
`isolated C)’ components is shown in Fig. 6.
`The CE irztgments and Bence Jones proteins
`formed reactions of nonidentity; absorption of
`the antiserum with one of the intact Bence
`
`jones proteins left precipitin reactivity with
`
`the C)’ components. The constant region of the
`x light chain (C94) contains :1 ‘hidden’ antigenic
`determinant that is revealed only after pepsin
`cleavage of the x chain into V,,- and CL—related
`components or by unfolding the intact 2 chain
`with a dissociating solvent (26). The reactivity
`of C: Wms, Oak, and Edw and C1’: Len was
`tested with an anti—x Bence Jones antiserum
`(anti—;¢ jo) that has specificity for the ‘hidden'
`C,‘ antigenic determinant. The specificity of
`this antiserum for CF Len is evident
`in the
`imniunoelectrophoretic
`analysis
`depicted in
`Fig. 7. However, the antiserum did not distin-
`
`guish between the Bence _]ones proteins and
`any of the three C: components. Absorption
`oi‘
`the antiserum with an intact
`:4 Cl'1aiI1
`re-
`
`moved precipitin reactivity with the CI’: com-
`poncnts and the Bence _]oncs proteins but not
`with the C)’ Len fragment.
`Comparaiive immunochcmical studies were
`also performed with two light chains possessing
`extensive
`internal V-region deletions
`(Table
`III). Kappa-chain Sac lacks ~70 amino acid
`residues, corresponding to positions '19 through
`88 in the VI_ (42), and )\-chain Sm lacks M81
`residues between positions 31 and 109 in the
`V,_ (15); both proteins, however, have intact
`C regions. On the basis of reactivity with an-
`ti—;¢, anti—i\, and anti—FII
`light—chain antisera,
`proteins Sue and Sm were readily classified
`
`

`
`Table III. Light chains with extensive V-region dele-
`Lions
`
`
`Present
`Missing
`Present
`
`xSac
`I
`18 [19
`88]
`89
`214
`
`ASm
`l—-——30 [31
`109] 110
`214
`
`as x— and }\-type, respectively. The precipitin
`reactions given by these two proteins were gen-
`erally comparable to those of complete Bence
`Jones proteins; With certain antisera, however,
`the intensity of reactions was notably less, es-
`pecially those given by protein Sm. The most
`striking differences were apparent with the an-
`tiscra to FII yG—globulin and to its Fab frag-
`ment. Proteins Sac and Sm reacted weakly with
`both of these antisera and were antigenically
`deficient when compared with intact x or A
`Bence Jones proteins. Although these antisera
`distinguish between x and A chains (see Fig. 6),
`it protein Sac and A protein Sm together formed
`a precipitin reaction of identity. Proteins Sac
`and Sm were also tested against the anti—light-
`chain antisera that have specificity for the ‘hid-
`den’ C2»: and CA antigenic determinants. With
`the anti—;-,- Jo antiserum, x protein Sac and a
`complete ;-: chain gave a precipitin reaction of
`identity, and both were antigenically deficient
`to a Cfi fragment. However, with the anti-A
`I-Iil antiserum, a complete A chain was antigeni-
`cally deficient to both the A protein Sm and a
`CPI fragment, which together formed a precip-
`itin reaction of identity.
`
`reactivity of VL—relatcd components
`The
`formed by peptic digestion of x and A Bence
`Jones protein was also tested against the anti-
`sera used to study CL—related components. The
`V‘:
`fragments were recognized only by anti-
`sera to the homologous Bence Jones protein and
`by certain antisera with specificity for Vl_—relat-
`ed antigenic determinants,
`such as V—region
`subgroup or isotypic antigenic determinants (25,
`45).
`The effect of light—chain conformation on ex-
`pression of x and A antigenicity was investigat-
`ed through immunochemical comparisons of
`Bence Jones proteins exposed to chemical agents
`capable of disassociating noncovalent and cova-
`lent bonds. Bence Jones proteins were dissolved
`
`Locali::an'on of H and A Arizigeaiie Deterinimzrits
`
`691
`
`
`
`Fig. 8. Irnmunodiffusion analysis of native, denatured,
`and renatured Bence Jones protein. The peripheral
`antigen wells contain a K Bence Jones protein in [I]
`0.02M phosphate-0.15M NaCl buffer, pH, 7.2; [2]
`6M urea-phosphate—saline buffer; [3] 0.1M 2-mercap—
`toethanol-phosphate-saline buffer; [4] 6M urea-0.1M
`2-Inercaptoethanol-phosphate-saline buffer; and [S]
`the urea-mercaptoethanol-treated protein dialyzed
`against phosphate-—saline buffer. The central well
`contained an antiserum prepared against the pepsin-
`derived Fab fragment of FII ‘(G-globulin.
`
`in a
`at a final concentration of 0.25 mg/ml
`0.02M phosphate-0.15M NaCl buffer, pH 7.2,
`and in a similar buffer containing either 6M
`urea or 0.1M 2—mercaptoethanol or a combina»
`tion of the two. No differences in prccipitin
`reactivity were evident among urea-treated,
`mercaptoethanol-treated,
`and
`native
`(phos-
`phate-saline) x and A Bence Jones proteins. On
`the other hand,
`the presence of both urea
`and mercaptoethanol markedly affected light-
`chain
`antigenicity.
`Proteins
`dissolved
`in
`phosphate—saline buffer containing both 6M
`urea and 0.1M 2-mercaptoethanol
`failed to
`form visible precipitin reactions with the anti-
`Fll light Chain, anti-Fll Fab, and anti-F11 ‘(G-
`globulin antisera, and reacted only weakly or
`not at all with antiseta prepared against
`the
`homologous or heterologous Bencc Jones pro-
`teins. However, the loss of x and A antigenicity
`was reversible; dialysis of the urea- and Iner-
`captoethanol-treated proteins, first against 4M
`urea, phosphate-saline buffer, pH 7.2, and then
`against
`the phosphate-saline buffer,
`restored
`precipitin reactivity of the renatured proteins
`
`(Fig. 8).
`
`

`
`692 A. Solomon
`
`DISCUSSION
`
`The reactivity of intact and incomplete Bence
`jones proteins and of naturally occurring and
`enzymatically produced fragments of Bence
`_]ones proteins, as compared by immunochemi-
`cal analyses with antisera specific for light-
`chain antigenic determinants, demonstrates the
`cssentiality of the intact light polypeptide chain
`for expression of the x and A. antigenic determi-
`nants. Differences in x and .\ antigenicity were
`most apparent with pepsin-derived CL-related
`fragments but were also evident with light
`chains containing the entire C region but which
`lacked extensive portions of the V region.
`The ability to cleave specifically light chains
`into V1; and CL-related fragments led to stud-
`ies designed to determine whether a property
`of the intact light chain is indeed a function of
`the whole molecule or whether it is a charac-
`
`teristic of only the V or C domain (20, 45).
`For example, the idiotypic and certain isotypic
`antigenicities of an intact
`light chain are ex-
`pressed by the isolated VL, whereas other iso-
`typic and allotypic specificities structurally lo-
`cated on the V.‘ and CL, respectively, require
`the intact polypeptide chain for their expression
`(24, 25, 46, 4?). The Inv(1), (2), and (3) allo-
`typic specificities of x chains involve the ami-
`no acid residues located at positions 153 and
`191 and the C x domain (29, 50); however, the
`serologic expression of these Inv antigenicities
`is lost upon peptic cleavage of the a: chain into
`V1: and Cl;
`fragments (49). Despite the fact
`that C}:
`fragments derived from Inv(1), Inv
`(1.2), and Inv(3) Bence Jones proteins contain
`virtually the entire C domain (residues 117
`through 214),
`these fragments are devoid of
`lnv activity.
`Determination of the Inv antigenicity of x
`chain Sac and the
`* components indicate the
`contributory role of the Vx domain in expres-
`sion of Inv antigenicity. On an equimolar ba-
`sis, the luv antigenicity of Inv(1,2) protein Sac
`was similar to complete Inv(1,2) x chains; the
`C: Wms and C: Oak proteins also had de-
`tectable Inv antigenicity but at a level 16- to
`32-fold less than that of the intact Inv(3) Bence
`Jones proteins Wins and Oalt
`(48, 49). The
`
`additional polypeptide present on the CEI com-
`ponents and 9: chain Sac also masked the ‘hid-
`den’ antigenic determinant detected on C}:
`fragments (26).
`The V domain also contributes to certain
`
`physicochemical properties that are expressed
`by the intact polypeptide chain. For example,
`the characteristic thermal properties of
`the
`light chain (33) (which are responsible for the
`classical Bence _]ones protein ‘heat
`test’) are
`manifested by the V,‘ (45). Each of the proteins
`that lacked all or part of the V region (that is,
`C‘]:
`fragments, CE components, x chain Sac,
`and A chain Sm) failed to precipitate on heat-
`ing, remaining soluble at 100°C. Comparison
`of the fluorescent and phosphorescent proper-
`ties of V,_ and C,‘ with those of the whole pro-
`tein showed that
`the luminescence of VL has
`many features in common with that of the in-
`tact light chain, whereas the C,‘ and particu-
`larly the CE possess markedly different fea-
`tures (23).
`Immunoglobulin molecules are characterized
`by a series of compact globulin domains linked
`by an extended (linear) peptide sequence (11).
`Comparative optical rotatory dispersion and
`circular dichroism spectral analyses of V1, C1,
`and intact light chains indicate that the V and
`C domains are each folded into a compact
`structure allowing relatively little, if any.
`in-
`terdomain interaction (6, 16, 19). However. a
`potential
`for
`interdomain interaction
`does
`exist: our studies of the luminescent properties
`of a large number of human light chains have
`shown that the orientation of the tryptophyl
`residue(s) in the V domain can indeed influence
`the tryptophyl-disulfide link interactions in the
`C domain (23). The loss of x and A antigenicity
`of urea
`and mercaptoethanol-treated light
`chains plus the restoration of antigenicity on
`renaturation demonstrates the conformational
`
`dependency of the 3: and A antigenic determi-
`nants, thus providing further evidence of inter-
`domain interaction.
`
`X—ray diffraction data derived from crystals
`of the human A Bence Jones protein dimer Mtg
`(35), the human A Fab’ fragment New (31, 32),
`and a murine pg Fab fragment (36) indicate rel-
`atively few intcrdomain interactions as com-
`
`
`
`

`
`pared with the intradomain interactions cre-
`ated by the V—V or C—C associations. One of
`the monomeric units (Monomer 2)
`in the A
`light-chain dimer Mcg (35) structurally shares
`features noted for other light chains (8, 31, 36).
`The closeness of the residues at positions 174
`and 82 in the C and V domains, respectively,
`in Monomer 2 (35) provides at least one site
`for potential steric interaction between light-
`chain domains. These positions are homolo-
`gous to the location of a disulfide linkage be-
`tween the C and V domains of certain rabbit x
`light chains (1, 51). Further, the residue cor-
`responding to position 174 occupies a complete-
`ly exposed location on x and A human and
`murine light-chain molecules (18), and the ty-
`rosyl residue at position 173 is the most readily
`iodinated of all the tyrosines in human x chains
`(39). The differences in expression of x or A
`antigenicity observed for the C11‘: fragments, CE
`components, and light chains Sac and Sm may
`reflect the fact that all of these proteins lack
`that portion of the V region that includes posi-
`tion 82.
`
`The fact that the ‘switch’ region between V
`and C domains also contributes to the expres-
`sion of x or A antigenicity is evidenced by the
`diversity in reactivity among the C: compo-
`nents and CE fragments. The ‘switch’ region,
`as well as other structurally defined regions
`responsible for
`isotypic and allotypic light-
`chain specificities, is on the surface of the mole-
`cule and is thus exposed to solvent
`(32, 35).
`The observed flexibility in the hinge region link-
`ing V and C domains may also permit addi-
`tional V—C interactions (8, 31, 32, 35, 36).
`Differences in the V—C bond angles could also
`account for the marked diversity in the suscep-
`tibility of human light chains to proteolytic
`cleavage into V,‘ and CL fragments (20, 45).
`Certain Bence Jones proteins are readily cleaved
`in their native state, whereas other proteins,
`especially A chains, are rendered more suscep-
`tible to cleavage by partial reduction and alky-
`lation (20, 45).
`The V domains and C domains of light
`chains and of heavy chains are encoded by sep-
`arate genes (17, 30). The V domain of x chains
`(Vx ) always associates with the C domain of Z
`
`Locafizarion of at and it Arrrigeuic Deternrirrarits
`
`693
`
`chains (Cu ) and, similarly, V1 always associ-
`ates with Cl ; hybrid molecules do not occur
`—that is, V” —C,\ , V)‘ —CK . In contrast, the
`V domain of heavy chains (VH) may associate
`with any of the C domains (CH) from each of
`the five classes of heavy chains;
`in fact,
`the
`transition from IgM to IgG (or IgA) antibody
`synthesis reflects the substitution of Cu regions
`and the preservation of the V1, region of both
`types of molecules (41, 54, 55). Structural char-
`acterization and hybridization kinetic analy-
`ses of immunoglobulin mRNA, which has been
`isolated from light-chaimsynthesizing murine
`plasma cell
`tumors, show a single integrated
`rnRNA molecule (22, 28). The contributory
`roles of the V domain, C domain, and the in-
`terdomain ‘switch’ region in expression of x and
`A antigenicity indicate that the 2: or A deter-
`minants are markers of an integrated V—C
`gene, suggesting that
`these determinants may
`serve as the recognition signal for V—C integra-
`tion. Variability in expression of the 'kappa' or
`the ‘lambda’ gene may account for the marked
`interspecies differences in the ratio of x to A
`light chains (17) and, additionally, account for
`alterations in the x to A ratio noted clinically
`(3, 5, 37, 57).
`
`ACKNOWLEDGEMENTS
`
`This study was supported by USPHS Research
`Grants CA 10056-11, CA 15173-02, and CA
`13237-04 from the National Cancer Institute.
`
`REFERENCES
`
`1. Appella, E., Roholt, O.A._. Chersi, A., Raclzimski,
`G. 8: Pressman, D. Amino acid sequence of the
`light chain derived from a rabbit anti-p-azoberr
`zoate antibody of restricted heterogeneity. Bio-
`chem. bfophys. Res. Cemmun. 53, 1122, 1973.
`2. Baglioni, C., Cioli, D., Gorini, G., Rufiilli, A. 8:
`Alescio-Zonta, L. Studies on fraynents of light
`chains of human imrnunoglobulins: genetic and
`biochemical implications. Cold Spr. Harb. Symp.
`quamt. Biol. 32, 147, 1967.
`3. Barandun, S., Morell, A., Skvaril, F. 8: Ober-
`dorfer, A. Deficiency of x- or 1-—type immuno-
`globulins. Blood 47, 79, 1976.
`Itnmunoglobulin
`4. Berggard,
`I.
`8: Peterson, P.
`components in normal human urine. p. 71 in
`Killandcr, J. (ed.) Gamma Globulins, Nobel Sym-
`
`
`
`

`
`694
`
`.4. Solomon
`
`posium 3. Al