Effect of Single Amino Acid Substitutions by Asn and Gln on Aggregation Properties of Bence-Jones Protein BIF.

The nature of renal amyloidosis involving Bence-Jones proteins in multiple myeloma is still unclear. The development of amyloidosis in neurodegenerative diseases is often associated with a high content of asparagine and glutamine residues in proteins forming amyloid deposits. To estimate the influence of Asn and Gln residues on the aggregation of Bence-Jones protein BIF, we obtained recombinant BIF and its mutants with the substitution of Tyr187→Asn (Y187N) in α-helix of CL domain, Lys170→Asn (K170N) and Ser157→Gln (S157Q) in CL domain loops, Arg109→Asn in VL-CL linker (R109N) and Asp29→Gln in VL domain loop (D29Q). The morphology of protein aggregates was studied at pH corresponding to the conditions in bloodstream (pH 7.2), distal (pH 6.5) and proximal renal tubules (pH 4.5) by atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS). The Lys170→Asn replacement almost completely inhibits amyloidogenic activity. The Y187N forms fibril-like aggregates at all pH values. The Arg109→Asn replacement resulted in formation of fibril-like structures at pH 7.2 and 6.5 while the substitutions by Gln provoked formation of those structures only at pH 7.2. Therefore, the amyloidogenic properties are highly dependent on the location of Asn or Gln.

Influence of a Single Point Mutation in the Constant Domain of the Bence-Jones Protein bif on Its Aggregation Properties.

Multiple myeloma nephropathy occurs due to the aggregate formation by monoclonal immunoglobulin light chains (Bence-Jones proteins) in kidneys of patients with multiple myeloma. The mechanism of amyloid deposit formation is still unclear. Earlier, the key role in the fibril formation has been assigned to the variable domains that acquired amyloidogenic properties as a result of somatic mutations. However, fibril formation by the Bence-Jones protein BIF was found to be the function of its constant domain. The substitution of Ser177 by Asn in the constant domain of the BIF protein is most likely an inherited than a somatic mutation. To study the role of this mutation in amyloidogenesis, the recombinant Bence-Jones protein BIF and its mutant with the N177S substitution typical for the known immunoglobulin Cκ allotypes Km1, Km1,2, and Km3 were isolated. The morphology of aggregates formed by the recombinant proteins under conditions similar to those occurring during the protein transport in bloodstream and its filtration into the renal glomerulus, in the distal tubules, and in the proximal renal tubules was analyzed by atomic force microscopy. The nature of the aggregates formed by BIF and its N177S mutant during incubation for 14 days at 37°C strongly differed and depended on both pH and the presence of a reducing agent. BIF formed fibrils at pH 7.2, 6.5, and 10.1, while the N177S mutant formed fibrils only at alkaline pH 10.1. The refolding of both proteins in the presence of 5 mM dithiothreitol resulted in the formation of branched structures.

A strategy for synthesis of pathogenic human immunoglobulin free light chains in E. coli.

Monoclonal immunoglobulin light chains are normally synthesized in excess compared to the heavy chain partners and can be detected in serum and urine ("free" LC). Occasionally free LC are per se cause of organ toxicity, as in free LC-related disorders. In AL amyloidosis, the most common of these conditions, free LC with peculiar biophysical properties related to their primary structure damage target organs and organize in amyloid fibrils. Unlimited availability of well-characterized free LC is instrumental to investigate the toxic effect of these proteins and to study their interactions with targets. We present a straightforward strategy to obtain recombinant monoclonal free LC by using a bacterial system. These proteins, expressed as inclusion bodies, were subjected to solubilization and refolding procedures to recover them in native form. To minimize differences from the circulating natural LC, full-length recombinant LC were expressed, i.e. complete of variable and constant regions, with the original amino acid sequence along the entire protein, and with no purification tags. The strategy was exploited to generate free LC from three AL amyloidosis patients. After purification, recombinant proteins were biochemically characterized and compared to the natural Bence Jones protein isolated from one of the patients. Results showed that the recombinant free LC were properly folded and formed homodimers in solution, similar to the natural Bence Jones protein used for comparison. Furthermore, as proof of pathogenicity, recombinant proteins formed amyloid fibrils in vitro. We believe that the present strategy represents a valuable tool to speed research in free LC-related disorders.

[The appearance of t(9;22)(q34;q11.2) in BJP-λ type multiple myeloma during maintenance therapy including lenalidomide].

A 61-year-old man, who was diagnosed with Bence-Jones protein (BJP)-λ type multiple myeloma, was treated with bortezomib. Although maintenance therapy including lenalidomide was continued, t(9;22)(q34;q11.2) was detected in the marrow cells by a cytogenetic study. The increased incidence of a secondary malignancy after treatment with lenalidomide for multiple myeloma has been highlighted in previous clinical trials; however, reports on the presence of t(9;22)(q34;q11.2) or the onset of chronic myelogenous leukemia have yet to be found. Although the cause of chronic myelogenous leukemia with the lenalidomide treatment is not yet clear, it is an interesting case.

[The relation between Bence Jones protein gene polymorphism and function of renal tubular-epithelial cell in multiple myeloma].

OBJECTIVE: Bence Jones protein (BJP) plays an important role in multiple myeloma (MM) renal lesion, we try to study the relation between the characteristics of BJP variable gene and the function of renal tubular-epithelial cell (TEC). METHODS: MM patients whose function of TEC was abnormal at diagnosis constituted a group damage and patients whose function of TEC was normal for a long period a group normal. We also collected MM patients and divided them into a group BJPkappa and a group BJPlambda. Total RNAs were isolated from the mononuclear cells of bone marrow and reverse transcription was carried out with an oligo dT18 primer; these cDNAs were then amplified with PCR, cloned and sequenced, the comparison and analysis of the sequences were made according to current ESBC/Gen Bank sequence directories. RESULTS: 3/5 cases of BJPlambda use V3-4 gene in the group damage, the replacement R mutation ratio of CDRS in the group damage (7.57 +/- 3.40) was higher than that in the group normal (4.25 +/- 1.90) and higher than that of FWRS in the group damage (3.29 +/- 1.25); R mutation ratio of CDRS region in the group BJPlambda (6.64 +/- 2.38) was higher than that in the group BJPkappa (4.10 +/- 2.13) and higher than that of FWRS in the group BJPlambda (2.91 +/- 0.94), R mutation ratio of CDRS both in the group BJPkappa or BJPlambda was higher than that of FWRS respectively. CONCLUSIONS: The higher ratio of renal lesion in some MM patients and BJPlambda patients is correlated with the high ratio of their variable gene R mutation which result in changing organization and physicochemical activity of BJP. BJP translated by some subtype genes may incline to injure the function of TEC.

Improvement of catalytic antibody activity by protease processing.

An immunoglobulin L chain (HIR) was treated with lysyl-endopeptidase. Gel filtration chromatography of the digestion mix identified a peak displaying a significantly higher specific catalytic activity than that of the original sample. The protein in the peak was 11 kDa in size and constituted the VL fragment of HIR. The Km and Kcat values of Chromozym TRY hydrolysis for HIR were 1.5 x 10(-4) M and 6.2 min(-1), and for the VL fragment 7.3 x 10(-4) M and 4.8 x 10(2) min(-1), respectively. Three out of the five BJPs studied in this paper displayed elevated catalytic activity after processing with lysyl-endopeptidase. Similar results were also obtained for the complete antibody.

Dealing with intractable protein cores: protein sequencing of the Mcg IgG and the Yvo IgM heavy chain variable domains.

The VH domains of two human monoclonal antibodies, designated Mcg IgG1(lambda) and Yvo IgM(kappa), were particularly intractable to standard protein sequencing protocols. Peptides liberated from the VH domains of these proteins, using standard enzymatic or chemical cleavages, invariably precipitated during the procedures. Boiling in SDS containing buffers dissolved precipitates and the peptides were separated using SDS-PAGE. Fully overlapped VH sequences were obtained with a series of 'in-gel' cleavages, followed by passive/differential transfers of peptides onto PVDF membranes. Both the in-gel cleavages and passive transfers could be applied to 'wet' or 'dry' gels so that gels could be archived and used at a later date to obtain additional sequence information from a fragment of interest. Repetitive yields of even the most insoluble peptides were such that the sequences of various peptides from relatively complex mixtures of peptides could be assigned with confidence. Despite the overall success of the sequencing, we occasionally referred to electron density maps, calculated for crystals of the Fab of Yvo IgM, to resolve particular sequences and confirm ambiguous amino acid assignments. Methods we describe in this report should be generally useful for obtaining sequences of proteins with intractable cores and may find many applications in the 'post genomic era'.

Unraveling of the polymorphic C lambda 2-C lambda 3 amplification and the Ke+Oz- polymorphism in the human Ig lambda locus.

Two polymorphisms of the human Ig(lambda) (IGL) locus have been described. The first polymorphism concerns a single, 2- or 3-fold amplification of 5.4 kb of DNA in the C(lambda)2-C(lambda)3 region. The second polymorphism is the Mcg(-)Ke(+)Oz(-) isotype, which has only been defined via serological analyses in Bence-Jones proteins of multiple myeloma patients and was assumed to be encoded by a polymorphic C(lambda)2 segment because of its high homology with the Mcg(-)Ke(-)Oz(-) C(lambda)2 isotype. It has been speculated that the Mcg(-)Ke(+)Oz(-) isotype might be encoded by a C(lambda) gene segment of the amplified C(lambda)2-C(lambda)3 region. We now unraveled both IGL gene polymorphisms. The amplification polymorphism appeared to result from a duplication, triplication, or quadruplication of a functional J-C(lambda)2 region and is likely to have originated from unequal crossing over of the J-C(lambda)2 and J-C(lambda)3 region via a 2.2-kb homologous repeat. The amplification polymorphism was found to result in the presence of one to five extra functional J-C(lambda)2 per genome regions, leading to decreased Ig(kappa):Ig(lambda) ratios on normal peripheral blood B cells. Via sequence analysis, we demonstrated that the Mcg(-)Ke(+)Oz(-) isotype is encoded by a polymorphic C(lambda)2 segment that differs from the normal C(lambda)2 gene segment at a single nucleotide position. This polymorphism was identified in only 1.5% (2 of 134) of individuals without J-C(lambda)2 amplification polymorphism and was not found in the J-C(lambda)2 amplification polymorphism of 44 individuals, indicating that the two IGL gene polymorphisms are not linked.

The V kappa III subgroup light chain proteins in AL amyloidosis & autoimmune diseases.

BACKGROUND & OBJECTIVES: Light chain associated amyloidosis (AL) is characterized by extracellular deposition of immunoglobulin light chain and its fragments. In vitro and in vivo studies have shown that some light chains are nonamyloidogenic and nonnephrotoxic, whereas others are potentially amyloidogenic. Some light chains are prone to be deposited as rheumatoid materials, and also as nodular amorphous aggregates (light chain deposition diseases). These findings suggest that specific sequence element(s) may control the various kinds of light chain associated diseases. In this study we tried to identify such sequence element(s). METHODS: Two Bence Jones proteins (BJPs), NIG93 and NIG2 of subgroup V kappa III, were characterized and compared with other members of the same subgroup whose sequences are available in the data base. RESULTS: Both NIG93 and NIG2 proteins had sequences characteristics of V kappa IIIa as distinguished from V kappa IIIb, subsubgroup proteins. They also contained several novel substitutions, such as Met-37, Leu-40, Val-58, and IIe-85 in NIG93, and Val-2, His-29, Arg-50, and Ile-72 in NIG2. The data accumulated at present indicate that all members of the V kappa IIIa subsubgroup are related to either AL amyloidosis or rheumatoid arthritis, whereas the V kappa IIIb proteins are related to autoimmune diseases. INTERPRETATION & CONCLUSION: These observations indicate that subgroup-specific residues might be critical for light chain pathogenesis, at least for the V kappa III proteins. Point mutations within these proteins may be another structural element controlling their conformation as well as their pathogenic aggregation.

Structural relationship of kappa-type light chains with AL amyloidosis: multiple deletions found in a VkappaIV protein.

Two amyloidogenic Bence Jones proteins (Am37 VkappaIV and NIG1 VkappaI) and one non-amyloidogenic protein (NIG26 VkappaIII) were characterized. The protein Am37 had four deletions when compared with the translated germ-line gene sequence: two Ser residues following position 27 (27e, 27f) in CDR1 and two amino acids Pro-44, and Tyr-49 in FR2 were deleted. A strictly conserved salt-bridge-forming amino acid, Asp-82, was replaced by the hydrophobic residue Leu. In a comparative study of amyloidogenic and non-amyloidogenic proteins, five amino acids (Ser-10, Ala-13, Ser-65, Gln-90, and Ile-106) were found to be unique to NIG1 and several other amyloidogenic proteins. Additional substitutions also occur within these proteins. These substitutions might be significant in altering protein folding as well as in contributing to their aggregation as amyloid fibrils.

Molecular structure of the amyloid-forming protein kappa I Bre.

The molecular structure of the amyloid-forming Bence-Jones protein kappa I Bre has been determined by X-ray crystallography at 2.0 A resolution. The fragment from the kappa chain of immunoprotein contains 107 amino acid residues, and polymerizes in the crystal form into a giant helical spiral, surrounding a cylinder of water 50 A in diameter with a repeat of 77.56 A, containing 12 kappa molecules, plus another 12 molecules from neighboring parallel spirals. The resulting structure has many features which have been found or suggested from studies on the protein fibrils found in amyloid deposits. From the results of the X-ray crystal structure a hypothesis is presented for the structure and formation of the amyloid fibril.

Characterization of a light chain product of the human JC lambda 7 gene complex.

The human light chain JC lambda locus is comprised of seven distinct segments, designated JC lambda 1, JC lambda 2, JC lambda 3, JC lambda 4, JC lambda 5, JC lambda 6, and JC lambda 7. Whereas three of these seven represent pseudogenes (psi Clambda 4, psi C lambda 5, and psi C lambda 6), the JC lambda 1, JC lambda 2, and JC lambda 3 complexes are functional, as demonstrated by the finding of their protein products through sequence analyses of lambda-type Bence Jones proteins and light chains derived from monoclonal Igs. Although the JC lambda 7 segment also appears functional, as evidenced through analysis of lymphocyte-derived mRNA, heretofore no monoclonal JC lambda 7-containing lambda-chains have been identified. Serologically, two distinct isotypic markers, Mcg and Oz, are associated, respectively, with JC lambda 1 and JC lambda 3 proteins, in contrast to JC lambda2 components, which do not express these determinants and represent a third isotype. Although another serologic marker, Ke (Kern), considered a fourth isotype, has been assigned to the JC lambda 7 complex, this relationship has been questioned. We now report the primary structural features of a lambda-type Bence Jones protein that include the four distinctive residues encoded by the JC lambda 7 gene segment. This protein, obtained from a patient with multiple myeloma and designated MCP, represents the first example of such a molecule and provides definitive evidence that the JC lambda 7 gene complex is functional. Additionally, comparison of the C lambda sequences of Mcg-/Oz- Bence Jones proteins MCP and KERN supports the contention that the Ke-associated one-residue amino acid variation at position 152 reflects a C lambda A2 polymorphism and that yet another isotypic marker, provisionally designated Mcp, is encoded by the JC lambda 7 gene segment. Thus, we posit that there are four human JC lambda isotypes, Mcg, Ke-Oz-/Ke+Oz-, Ke-Oz+, and Mcp, that represent, respectively, products of the JC lambda 1, JC lambda 2, JC lambda 3, and JC lambda 7 gene complexes.

Overrepresentation of the V kappa IV subgroup in light chain deposition disease.

The variability subgroup of human monoclonal kappa chains purified from urine in 3 consecutive patients with myeloma associated light chain deposition disease was determined from amino acid sequences of their first framework regions (FR1). N-glycosylation was searched for by N-glycosidase F treatment. These data together with our previously published results, indicate the pathogenic potential of the rare V kappa IV subgroup and confirm the absence of detectable serum and urine free monoclonal light chains when they are N-glycosylated.

Proteolytic excision and in situ cyclization of a bioactive loop from an REI-VL presentation scaffold.

Covalent cyclization of peptides is an important tool in structure-function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor-bound conformation. Previously we described a 2-step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N-terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533-539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo-LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI-VL) with an RGD-containing sequence engineered into its CDR3 and flanked by Lys residues. This REI-VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor alpha IIb beta 3. The RGD-containing peptide excised by endo-LysC from the REI-VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, and IC50 of about 10 nM. Significantly, both the N alpha-acylation and the cyclization reactions occur efficiently even in the context of the other endo-LysC fragments of REI-VL, which suggests that the reaction may prove useful in converting mixtures of endo-LysC products of many proteins into the corresponding cyclic peptides in situ.

Dimerization of Bence Jones proteins: linking the rate of transcription from an Escherichia coli promoter to the association constant of REIV.

Homodimers of immunoglobulin VL domains are minimal models of antibodies in that they display an ensemble of six hypervariable loops. Bence Jones protein REI is a mixture of a complete kappa light chain and the corresponding variable domain (REIV). The known three-dimensional structure of the REIV dimer (Epp et al., 1975, Biochemistry 14, 4943-4952) provides a basis for studying dimer stabilization by protein engineering. Mutant REIV-L94H was constructed and shown to have an equilibrium constant of dimerization about one order of magnitude higher than wildtype REIV. By fusing REIV and variants to the aminoterminal part of the Vibrio cholerae ToxR regulator protein (Miller et al., 1987, Cell 48, 271-279), a transcriptional signal in E. coli can be derived from REIV homodimer formation constant. The system senses dimerization of the immunoglobulin part of the fusion protein, located in the periplasmatic space, and transduces the signal as transcriptional activation to a ctx::lacZ gene construct integrated into the E. coli chromosome. There is positive correlation between the propensities of homodimer formation and the rate of transcriptional initiation at the ctx promoter. Since beta-galactosidase levels can easily be measured colorimetrically in crude cell lysates of a large number of clones using an ELISA reader, this procedure constitutes all elements required for a genetic screen in E. coli for immunoglobulin variants with altered association constants.

Primary structure of a variable region of the V kappa I subgroup (ISE) in light chain deposition disease.

Although structural abnormalities of monoclonal immunoglobulin light chains (LC) are suspected to play a determinant role in non-amyloid light chain deposition disease (LCDD), this condition is as yet poorly documented at the molecular level, since only three sequences have been reported to date. In a case of myeloma-associated LCDD, the patient's urine contained an unglycosylated kappa Bence Jones protein made up of dimers and monomers with an apparent molecular mass of 25,000 which was assigned to the V kappa I subgroup by N-terminal amino acid sequencing. The complete variable region sequence of the monoclonal kappa chain produced by the malignant plasma cells was amplified by polymerase chain reaction (PCR) using small amounts of material obtained by bone marrow aspiration. The sequence of three independently amplified cDNA clones derived from a normal-sized kappa messenger RNA was identical to that of the urinary kappa chain. The kappa mRNA had an overall normal structure made up of a V kappa I sequence rearranged to J kappa I. Several unusual features of the variable region (the first complete V kappa I sequence reported in LCDD) included three substitutions that introduced hydrophobic residues at spatially close positions. The strategy associating N-terminal sequence determination and cDNA cloning by PCR could help in accumulating new sequence data and improving our understanding of LCDD pathogenesis.

General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli. Production of the Bence-Jones protein REIv via fusion to beta-lactamase.

A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli. This procedure, which has been applied to the production of the VK domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with beta-lactamase as the amino-terminal fusion partner. The beta-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure. The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage. After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture. A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops. It exploits a colony color genetic screen and can be applied to any DNA sequence replacement. Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide.

Immunologic studies in identical twins concordant for juvenile rheumatoid arthritis but discordant for monoclonal gammopathy and amyloidosis.

Identical twins concordant for juvenile rheumatoid arthritis but discordant for monoclonal gammopathy and amyloidosis were the subjects of a study done with mixed leukocyte culture, anti-idiotypic antisera against serum and urinary M component from the amyloid-affected twin, and in vitro estimations of M-component idiotype synthesis by peripheral blood mononuclear cells. Immunohistochemical analysis of renal amyloid deposits in the affected twin showed AL amyloid of the lambda-II variable region subgroup. M-component idiotypes were confined only to the twin with serum and urine M components.

Serologically defined V region subgroups of human lambda light chains.

The availability of numerous antisera prepared against lambda-type Bence Jones proteins and lambda chains of known amino acid sequence has led to the differentiation and classification of human lambda light chains into one of five V lambda subgroups. The five serologically defined subgroups, V lambda I, V lambda II, V lambda III, V lambda IV, and V lambda VI, correspond to the chemical classification that is based on sequence homologies in the first framework region (FR1). Proteins designated by sequence as lambda V react with specific anti-lambda II antisera and are thus included in the V lambda II subgroup classification. The isotypic nature of the five V lambda subgroups was evidenced through analyses of lambda-type light chains that were isolated from the IgG of normal individuals. Based on analyses of 116 Bence Jones proteins, the frequency of distribution of the lambda I, lambda II/V, lambda III, lambda IV, and lambda VI proteins in the normal lambda chain population is estimated to be 27%, 37%, 23%, 3%, and 10%, respectively. This distribution of V lambda subgroups was comparable to that found among 82 monoclonal Ig lambda proteins. Considerable V lambda intragroup antigenic heterogeneity was also apparent. At least two sub-subgroups were identified among each of the five major V lambda subgroups, implying the existence of multiple genes in the human V lambda genome. The V lambda classification of 54 Ig lambda proteins obtained from patients with primary or multiple myeloma-associated amyloidosis substantiated the preferential association of lambda VI light chains with amyloidosis AL and the predominance of the normally rare V lambda VI subgroup in this disease.