Sound. 3. ImrmmeI., Vol. 5, 1976.
`
`Bence Jones Proteins and Light Chains of
`Immunoglobulins
`
`XIV. Conformational Dependency and Molecular Localization of the Kappa
`(K) 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. }. Inmzmwl. 5, 685-695, I976.
`
`The region on the light chain molecule responsible for expression of the it and ).
`antigenic determinants was determined by comparative imrnunochetnical analyses
`of intact Bence Jones proteins and naturally 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 1 antigenic determinants
`was also evidenced by denaturation-renaturation studies on x and ). chains. The
`V domain, C domain, and interdornain ‘switch’ region contribute to the expres-
`sion of 1-: or 1 antigenicity and to certain isotypic and allotypic specificities.
`
`A. Salomon, University of Tennessee Memorial Research Center. Knoxtriik. TN
`37920, USA.
`
`Immunochemical and structural analyses (9,
`13, 27, 34) of Bence Jones proteins, myeloma
`proteins,
`and Waldenstrfim macroglobulins
`have provided evidence that the light polypep-
`tide chains of all
`immunoglobulins consist of
`two chemically distinct types designated kappa
`(ac) 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 ~10? residues of variant sequence
`(VI) and a carboxyl-terminal portion of c\a107
`residues of constant sequence (CL). The VL and
`CL are under separate genetic control (17) and
`exist as two compact domains (designated V
`and C,
`respectively)
`linked by an extended
`cxnl0—residue section of the light polypeptide
`chain termed the ‘switch’
`region (31, 35). A
`given light chain may be distinguished as x or
`
`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 x or
`A antigenic determinants has not been estab-
`lished; it is not known whether these determi-
`
`nants rcside on the CL or V1‘ or whether their
`expression requires the intact light polypeptide
`chain.
`
`The relation between immunoglobulin struc-
`ture and antigenicity has been established
`through studies of the naturally occurring or
`enzymatically derived fragments and the chem-
`ically
`produced
`subunits
`oi
`homogeneous
`immunoglobulins (Bence Jones proteins, mys-
`loma. proteins, and Waldenstrom macroglobu-
`lins)
`characteristically found in association
`with multiple myeloma and related plasma cell-
`lymphocyte dyscrasias (43). The identification
`and characterization of urinary low-molecular
`
`SANOFI v. GENENTECH
`SANOFI V. GENENTECH
`IPR2015-01624
`IPR2015-01624
`EXHIBIT 2089
`EXHIBIT 2089
`
`
`

`
`536 A. Solomon
`
`weight proteins antigenically related to the V
`or C regions of Bence Jones proteins (2, 4, 7,
`IO, 44, 48, 52, 53, 56), the availability of un-
`usual Bence Jones proteins with extensive V re-
`gion deletions (15, 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 ac and A 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 24-11
`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 Visking 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 x chain Sac (42) were furnished by Dr. Al-
`len B. Edmundson, Dr. Elliott F. Osserman.
`
`respectively. The
`and Dr. Dorothy M. Parr,
`methods used for the preparation and isolation
`of light chains and of the papain-derived Fab
`fragment of PH 7G-globulin were as described
`previously (45).
`The Bence Jones proteins were cleaved by
`pepsin into constant half-related (CE ) and va-
`riant half-related (VE)
`fragments (45). The
`digestions were performed with an enzyme to
`protein ratio of 1:100 in 0.05M glycine—HCl
`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).
`lmmunoebemical 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 FII
`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 lnv 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 V,‘ or the CL (48).
`The new components, designated C;‘_, were
`found to be structurally and antigenically 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 CL-related fragment derived in vitro
`by peptic cleavage (CE) of the light chain (48).
`The amino-terminal sequences of CE isolated
`from urine specimens of Patients Wms, Oak,
`and Edw and the C11: prepared by pepsin cleav-
`
`Table I. In vivo (Cf) and in vitro (CE) constant—half—
`related fragments of it Bence Jones proteins
`Amino-terminus
`
`Protein
`
`Cf Wms
`CI’: Oak
`Cf Edw
`Ci’. Len
`
`Residue
`
`Positioni-
`
`Asp
`Thrflle
`Val
`Ile
`
`92
`97f106'["|'
`110
`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 9'?
`and 106, respectively.
`
`

`
`Lacah'zan'a1t of it and it Armlgeni‘.-: Determirtants
`
`687
`
`Table II. Amino-terminal sequences of it Bence Jones protein fragments
`
`92
`
`100
`
`‘WC
`
`III)
`
`I20
`
`
`
`CE wms Asp lle Phe Pro Gly Thr Phe (Ely Gln Gly Thr Lys Val Glu Ile L3.rsArgThrVal AlaA|'.IPrt>SerVulPheIle Phe Pro Pro
`‘f
`
`CE Oak 1
`Phe Gly
`cg; Oak 2
`Cf: Edw
`
`CE Len -—-j
`1" The solid lines indicate sequence identity to C* Wrns.
`
`age of Bence Jones protein Len were deter-
`mined by Dr.
`Donald Capra. The amino-
`terminal residues and sequences of the Cl‘: 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-
`noelectrophotetic analyses of urine specimens
`Wins, Oak, and Edw (containing both C: 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 -/_ Bence Jones pro-
`teins were utilized for
`these analyses. With
`anti-homologous protein antisera, the CE com-
`ponents and the CE fragment were antigeni-
`
`cally cleficient as compared with the intact
`Bence Jones protein (Fig.
`1); absorption of
`these antisera with a heterologous J-C chain left
`reactions only with the homologous
`intact
`Bence Jones proteins. Striking differences in the
`reactivity of the C[_—related components were
`found with the anti—heterologous x chain anti~
`sera. Certain antiscra reacted weakly or not at
`all with the CI’: components and the CE frag-
`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. I. With the anti-;,: Isr antiserum,
`the CE components and the CE fragment reac-
`ted with equal intensity, each forming a precip-
`itin reaction of identity with the intact Bence
`Jones protein. However, the anti—;: Bre antise-
`rum did not react at all with C{’_ Len and reac-
`
`WMS
`
`EDW
`
`LEN
`
`- nnI1-homo! BJF’
`
`' CInI1‘KlsI
`
`— - 0I'1|1‘KBl'£
`
`111
`
` 1
`
`1
`
`if
`
`Q
`
`1
`
`1
`
`cf EJP
`
`cf
`
`BJF
`
`saw
`
`cf
`
`c[
`
`3.49 v[
`
`l. Irnmunoelectrophoreric analyses of intact polypeptide chains and related fragments of four it Hence
`Fig.
`Jones proteins. The antigen wells under the designations WMS, OAK, and ED W" contained urine specimens
`from Patients Wrns, Oak, and Lidw, respectively; the antigen wells under the designation LEN contained a
`sample of Bence 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-fioiriol. B_‘iP) and against antisera
`prepared to heterologous it Bence Jones proteins Isr and Bre {anti-it Isr and anti-it Bra). B_‘fP : Bence Jones
`protein; C: = constant-half-related light-chain component found in vivo; Cf and VI’?
`; constant-halll and
`variant-ha1f—related light-chain fragments, respectively, produced in vitro by pepsin proteolytie cleavage of
`Bence Jones protein. The anode was located to the left.
`
`
`
`

`
`683
`
`.4 .
`
`.S'o}oJ-um:
`
`K Wms
`
`KJO
`
`Klsr
`
`xoak
`
`KEdw
`
`K Len
`
`anti - KBre
`
`Tig. 2. Imrnunoditfusion analysis of six >: llenee Jones
`proteins. The peripheral antigen wells contained six
`different anti-V. Hence Jones proteins {:4 Writs. X Oak.
`7.
`Ifdti‘.
`if. Len.
`it Isr. and 3-! _7m. The central anti-
`serum well contained an antiserum prepared against
`:«: Hence Jones protein lire (tmtf-‘/_ Brc).
`
`ted only weakly with Cl‘: Edw and CE Oak.
`but :1 strong reaction occurred with CE Wins.
`The anti-x Bre antiserum was a potent anti-:4
`antiserum;
`the prccipitin t‘L".1Ctl0I‘lS olzitained
`with six ;¢ Bence _]ones proteins are shown in
`the in1n1unoCliffusion analysis depicted in Fig.
`2. Differences in the reactivity among the CE
`components were evident with many other
`.tnti—.i-.--chitin antisera; generally, the intensity of
`
`WMS
`
`OAK
`
`precipitin reactions among the C,‘-related frag-
`ments followed the order: C?‘ Wnis 73> C It Oak
`.7“ Cl’: Edw > C}: Len.
`Other types of antiscr-.1 with specificity for
`5-! antigenic determinants also showed this same
`
`pattern of reactivity with the Clgrelated com—
`ponents, as illustrated in Fig. 3 by the reactions
`obtained with ttntisera to intact Fll yG-globu-
`lin and its subunits. The anti-Fl} light chains.
`anti-FII Fab, and antiAFll y(_}-globulin nntisera
`did not form visible prccipitin reactioiis with
`CE Len, and reactetl only weal-(ly with CF
`Edw and C: Oak;
`-.1 more intense precipitin
`reaction occurred with CE Writs. All
`three
`antisera reacted strongly with intact .1: (as well
`as intact A.) chains and, further, had the capac-
`ity to distinguish between 3 and .\ light chains.
`The results of
`immunodii-fusion analysis of
`three 2: and three A Bence Jones proteins with
`the anti—Fll Fab antiserum are shown in Fig. 4.
`We li-aye not as yet detected C:-type com-
`ponents in urine specimens from our patients
`excreting A Bence Jones proteins. For this rea-
`son we treated six different A Bence Jones pro-
`teins with pepsin at 37 ‘C or at 55"C (40) to
`generate
`components in an effort to test the
`reactivity of our anti—i\ chain antisera with CL-
`related fragments. The reactivity of intact A
`
`
`
`‘ uni: ' homo! 8.)?
`
`~ nnli-FII L'chmrI'.~
`
`- anti-F 3 Feb
`
`- onh-FE
`
`cf sap
`
`cf
`
`asp
`
`sap
`
`l
`
`C
`
`BJP
`
`a
`VL
`
`Immunoelectrophoretic analyses of intact polypeptide chains and related fragments of [our 2!. Hence
`Fig. 3.
`_l ones proteins. The antigen wells under the designations l"MS. ()rlK_. and l:'Dl.l"' contained urine specimens
`from Patients '06-'ms. Oak, and Edw. respectively; the antigen wells under the designation L1:'.’\-' contained a
`sample of licence Jones protein Len subjected to limited pepsin 1:-roteolysis at 37 ‘C. Each sample was tested
`against an antiserum prepared to the hornologous Bence Jones protein (aimllmmoi. 3'71"") and against antisera
`prepared to the light chains of F1] 7G-globulin (anti-I"II I.-chairzsj, the papain-derived 1-‘ah fragment of F1]
`- constant-
`-{G-globulin (mitt-}"II Fab), and to F11 ‘(G-globulin (ar.ru'~FU). Bj’P -- Hence Jones protein; ('I;_
`half-related light-chain component found in vivo; (IE and
`= eonstant—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 lelt.
`
`
`
`

`
`Localization of X. and ). zlririgerzic Determinants
`
`689
`
`Kwms
`
`G)
`
`G)
`
`—onri- nmcq
`
`Pepsin Dunes!
`ts5°cmuco
`
`_
`
`- anh- Fn L'ChflIl\§
`
`_
`
`r
`""'
`
`H L
`'
`
`‘
`
`).Hil
`
`(Len
`
`».CIe
`
`anti— FII Fab
`
`Fig. -1. Irnrnunodiffusion analysis of three 1’. and three
`}. Bence Jones proteins. The peripheral antigen
`wells contained three :2. Bence Jones proteins (:4
`Writs, 7. Eda-, and 1»: Len) and three 3'. Bence Jones
`proteins O. Meg,
`1. Cle, and It H17). The central
`well contained an antiserum prepared against
`the
`papain-derived Fab fragment of FIT 7G-globulin
`(anti-PI] Fab).
`
`Bence Jones protein Meg and CE Meg (formed
`by partial peptic proteolytic cleavage of pro-
`tein Mcg) is presented in Fig. 5. This immuno-
`electrophoretic analysis shows that the antise-
`rum prepared to Bence Jones protein Meg reac-
`
`1
`T
`HJF
`cf
`intact
`Immunoelectrophoretic analysis of
`:>.
`Fig.
`polypeptide chain and constant-half~relatcd fragment
`of a 1 Bence Jones protein. The antigen wells contain-
`ed a sample of 1 Benee Jones protein Meg subjected
`to limited pepsin proteolysis at SEC. The upper,
`middle, and lower antiserum troughs contained anti-
`sera prepared against 1 Hence Jones protein Meg
`(cmu'—}\. Meg), It Hence Jones protein Hil [mitt-7t Hil),
`and the light chains isolated from FII -_'G—globulin
`(mitt-FII Z.-chants), respectively.
`
`ted only with the intact protein and not with
`the CE component. The antiserum prepared to
`It Bence Jones protein Hil recognized both in-
`tact protein Meg and its CE, and, although not
`evident in the figure, the C E component form-
`ed a prccipitin reaction of nonidentity with
`the intact Bence Jones protein. None of 20 an-
`tisera prepared against other A Bence Jones
`proteins formed a visible precipitin reaction
`
`Imtnunodiffusion
`6.
`Fig.
`intact polypep-
`anlysis of
`tide chains and constant-
`
`of
`fragments
`half-related
`three 1+. Bence Jones proteins.
`The peripheral antigen wells
`in both patterns contained
`intact 7. Bence Jones proteins
`(1. Law, 1 Meg. and ). C113)
`and their 553C pepsin-de-
`rived
`constant-half-related
`
`fragments (CE Lev, Cf Meg,
`and CE C12). The central well
`in the pattern on the left con-
`tained an antiserum prepared
`against it Bence Jones pro-
`tein Hil
`(anti-1 Hit). The
`central well
`in the pattern
`on the right contained the
`same
`antiserum absorbed
`with intact 1 Benee Jones
`protein Lev(a:mt'-1 Ht? 2').
`
`
`
`Cp CI
`'-
`
`e
`
`_
`_
`Grill‘ is Hll
`
`"
`.
`.
`Clflll‘ A Hll A
`
`
`
`

`
`690 A. Solomon
`
`WMS
`
`ll
`
`- onti—:rJo 3
`
`- Unis -hornol. BJP
`
`- Ol'|il'lJU
`
`T
`
`l
`
`M
`
`T
`
`I
`
`In H
`
`cf a.iP
`
`c
`
`B-JP
`
`BJP
`
`cf
`
`c[
`
`a.iP V:
`
`Fig. 7. Immunoelectrophoretic analyses of intact polypeptide chains and related fragments of four Z Hence
`Jones proteins. The antigen wells under the designations ll5"M.S‘_. OAK. and EDI!” contained urine specimens
`from Patients \5C'r-ns, Oak. and Edw, respectively; the antigen wells under the designation LEN contained
`a sample of Bence 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 (ann'—lmmoI. BJP) and against an anti-
`serum prepared to ‘at Bence Jones prote_in Jo (aritiac 30}, and the anti-it Jo antiserum absorbed with a heterolo-
`gous X Benee Jones protein (anti-xjci /1). H71’ --—- Bence Jones protein; C}:
`: constant-half-related light-chain
`component found in vivo; (If and Vi:
`. constant-half— and variant-half-related light—chain components,
`respectively, produced in vitro by pepsin proteolytic cleavage of Bence Jones protein. The anode was to the
`left.
`
`formed from 5
`Mcg or with the
`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—FlI
`light chains, anti-FII
`
`Fab, or anti-Iill yG-globulin antisera. Usually,
`A chains are more resistant
`than 2 chains to
`
`cleavage by endopeptidases: specific proteoly—
`sis of A chains into VI, anti CL is facilitated by
`prior reduction and alkylation of the proteins
`(20, 45). Pcpsin cleaves x chains between posi-
`tions H6 and I17 in the C domain (38). Al-
`though the specific site of .\-chain cle-.1v-age by
`pepsin is still to be established. our own studies
`and other data (21) indicate that the sequences
`of CJ’ Fragments derived from A Bence Jones
`proteins include the entire C region and also
`extend -x-4 to ID residues into the V region.
`The anti-A Bence Jones protein I-Iii antiserum
`was unique in its capability to react with C].-
`related Fragments formed by pepsin cleavage of
`A chains. A comparative immunodiffusion anal-
`ysis of three A Bence Jones proteins and their
`isolated C1; components is shown in Fig. 6.
`The C11:
`iragnieiits and Bence Jones proteins
`formed reactions of nonidentity; absorption of
`the antiserum with one of
`the intact Benee
`
`Jones proteins left precipitin reactivity with
`
`the C? components. The constant region of the
`2: light chain (Cg) contains :1
`'hidden' antigenic
`determinant that is revealed only after pepsin
`cleavage of the it chain into V” and C1_—reIated
`components or by unfolding the intact 3 chain
`with a dissociating solvent (26). The 1‘E‘.‘lCtiVi'ty
`of CE Writs, Oak, and Edw and CE Len was
`tested with an anti—;¢ Bence Jones antiserum
`(anti—;¢ Jo) that has specificity for the ‘hidden'
`CL antigenic determinant. The specificity of
`this antiserum for CF Len is evident
`in the
`immunoclcctrophoretic
`analysis
`depicted in
`Fig. 7. However, the antiserum did not distin-
`
`guish between the Bence Jones proteins and
`any of the three C: components. Absorption
`of the '.1n[iSL‘t'uI'i'1 with an intact
`.2 chain re-
`
`moved precipitin reactivity with the Cl’: com-
`ponents and the Bence Jones proteins but not
`with the CJ’ Len fragment.
`Comparaiivc immunochemical studies were
`also performed with two light chains possessing
`extensive
`internal V-region deletions
`(Table
`III). Kappaechain Sac lacks m70 amino acid
`residues, corresponding to positions 19 through
`88 in the V,_ (42), and }t—chain Sm lacks M81
`residues between positions 3] and 109 in the
`VI. (15); both proteins, however, have intact
`C regions. On the basis of reactivity with an-
`ti-;¢, anti-A. and anti-FII light—chain antisera.
`proteins Sac and Sm were readily classified
`
`

`
`Table III. Light chains with extensive V-region dele-
`tions
`
`
`Present
`Missing
`Present
`
`xSac
`1
`18 [19
`88]
`89
`214
`
`)tSm
`1-:30 [31
`109] 110
`214
`
`as x— and A-type, respectively. The prccipitin
`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 it and A chains (see Fig. 6),
`2: protein Sac and A protein Sm together formed
`a prccipitin reaction of identity. Proteins Sac
`and Sm were also tested against the anti—light-
`chain antisera that have specificity for the ‘hid-
`den’ C:-: and CA antigenic determinants. With
`the anti—x Jo antiserum,
`:4 protein Sac and a
`complete :4 chain gave a prccipitin reaction of
`identity, and both were antigenically deficient
`to a Ca fragment. However, with the anti-A
`Hil antiserum, a complete A chain was antigeni-
`cally deficient to both the A protein Sm and a
`CPR fragment, which together formed a precip-
`itin reaction of identity.
`
`reactivity of VL—r-elated 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 V,_—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 imtnunochemical comparisons of
`Bence Jones proteins exposed to chemical agents
`capable of disassociating noncovalent and cova-
`lent bonds. Bence Jones proteins were dissolved
`
`Locafi.-:ati'ort of x and "A /lrztigeatfc Deterntiraants
`
`69]
`
`
`
`Fig. 8. Immunodifiusion analysis of native, denatured,
`and renatured Bence Jones protein. The peripheral
`antigen wells contain a K Bence Jones protein in [1]
`0.02M phosphate—0.15M NaCl buffer, pH, 7.2; [2]
`6M urea-phosphate—saline buffer; [3] 0.1M 2-mercap—
`toethanol—phospl1ate-saline bufier; [4] 6M urea-0.1M
`2-Inercaptoethanol-phosphate-saline buffer; and [S]
`the urea-rnercaptoethanol-treated protein dialyzed
`against phosphate-saline buffer. The central well
`contained an antiserum prepared against the papain—
`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 antong urea-treated,
`mercaptoethanol—treated,
`and native
`(phos-
`phate-saline) as 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 prccipitin reactions with the anti-
`FII light chain, anti-FII Fab, and anti-FII 7G-
`globulin antisera, and reacted only weakly or
`not at all with antisera prepared against
`the
`homologous or hetcrologous Bence Jones pro-
`teins. Howcver, the loss of :-c and A antigenicity
`was reversible; dialysis of the urea— and mer-
`captoethanol-treated proteins, first against 4M
`urea, phosphate-saline buffer, pH 7.2, and then
`against
`the phosphate-saline buffer,
`restored
`prccipitin 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
`Jones proteins, as compared by immunochemi-
`cal analyses with antisera specific for light-
`chain antigenic determinants, demonstrates the
`essentiality of the intact light polypeptide chain
`for expression of the is and A antigenic determi-
`nants. Differences in x and A 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 VL and CL, respectively, require
`the intact polypeptide chain for their expression
`(24, 25, 46, 47). The Inv(1), (2), and (3) allo-
`typic specificities of at 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 lnv antigenicities
`is lost upon peptic cleavage of the 2.: chain into
`V1: and CE fragments (49). Despite the fact
`that CE 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 C; components indicate the
`contributory role of the Vx domain in expres-
`sion of Inv antigenicity. On an equimolar ba-
`sis, the Inv antigenicity of Inv(1,2) protein Sac
`was similar to complete Inv(1,2) x chains; the
`CE Wins and CE Oak proteins also had de-
`tectable lnv antigenicity but at a level 16- to
`32-fold less than that of the-intact Inv(3) Bence
`Jones proteins Wms and Oak (48, 49). The
`
`additional polypeptide present on the CEI com-
`ponents and 9: chain Sac also masked the ‘hid-
`den’ antigenic determinant detected on C11:
`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 jones protein ‘heat
`test’) are
`manifested by the VL (45). Each of the proteins
`that lacked all or part of the V region (that is,
`C: fragments, C: components, 2: 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 CI‘ and particu-
`larly the CE possess markedly different fea-
`tures (23).
`lmmunoglobulin 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 linl-t interactions in the
`C domain (23). The loss of ac and A antigenicity
`of urea
`and mercaptoethanol-treated light
`chains plus the restoration of antigenicity on
`renaturation demonstrates the conformational
`
`dependency of the x and A antigenic determi-
`nants, thus providing furrher evidence of inter-
`domain interaction.
`
`X—ray diffraction data derived from crystals
`of the human A Bence Jones protein dimer Meg
`(35), the human A Fab’ fragment New (31, 32),
`and a murine x Fab fragment (36) indicate rel-
`atively few interdomain interactions as com-
`
`
`
`

`
`pared with the intradornain 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 7».-
`
`light chains (1, 51). Further, the residue cor-
`responding to position 174 occupies a complete-
`ly exposed location on :4 and A human and
`murine light-chain molecules (18), and the ty-
`rosyl residue at position 173 is the most readily
`ioclinated of all the tyrosines in human .2 chains
`(39). The differences in expression of X or A
`antigenicity observed for the CE 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 CI‘:
`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 VL 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 :4
`
`Localization of at and 3-. Ann'gem'c Determfrzants
`
`693
`
`chains (Cn ) and, similarly, V1 always associ-
`ates with C1 ; hybrid molecules do not occur
`—that is, V,‘ —Cl , V3‘ —Cx . In contrast, the
`V domain of heavy chains (V1,) may associate
`with any of the C domains (Cu) 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 VH 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-chain-synthesizing murine
`plasma cell
`tumors, show a single integrated
`mRNA 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 x 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, 0.A., Chersi, A., Radzimski,
`G. 6: Pressman, D. Amino acid sequence of the
`light chain derived from a rabbit anti-p-azoben-
`zoate antibody of restricted heterogeneity. Bia-
`chem. b:'ap}:_ys. Res. Commun. .53, 1122, 1973.
`2. Baglioni, C., Cioli, D., Gorini, G., Rutfilli, A. 6:
`Alescio-Zonta, L. Studies on fragments of light
`chains of human immunoglobulins: genetic and
`biochemical implications. Cold Spr. Harb. Symp.
`quant. Biol. 32, 147, 1967.
`8: Ober-
`3. Barandun, S., Morell, A., Skvaril, F.
`dorfet, A. Deficiency of x- or 1»-type immuno-
`globulins. Blood 47, 79, 1976.
`Immunoglobulin
`4. Berggérd,
`I.
`8: Peterson, P.
`components in normal human urine. p. 71 in
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`
`
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`
`694
`
`.4. Solomon
`
`posium 3. Almqvist and Wiksell, Stockholm,
`1967.
`.