Primary structure of the variable region of an amyloidogenic Bence Jones protein NIG-77

The complete amino acid sequence of the variable region of a Bence Jones protein NIG-77 from an individual with myeloma-associated systemic amyloidosis has been determined. This protein represents a complete light chain consisting of 216 residues and it has a sequence characteristic of V lambda I subgroup, which is closely homologous to that of another amyloidogenic V lambda I Bence Jones protein NIG-51, differing by 20 of 111 residues (82% homology). In contrast, it differs by 29 residues (74% homology) to that of non-amyloidogenic V lambda I light chain NIG-64. This finding shows that, in accordance with our previous report(1), the V lambda I-related light chains can further be divided into two distinct subsubgroups, V lambda I-1 and V lambda I-2, and the latter property seems to be more prone in association with the amyloid process.     

Amino acid sequence of the variable region of the light (lambda) chain from human myeloma cryoimmunoglobulin IgG Hil.

We have determined the complete amino acid sequence of the variable region of the light (lambda) chain from a human myeloma cryoimmunoglobulin (IgG Hil), the Fab fragment from which has been previously crystallized. The presence of unblocked alpha-amino terminal residue and the isolation of a CNBr fragment starting at position 46 and of a maleylated tryptic fragment spanning residues 61 to 189 provided three suitable starting points for automatic Edman degradation. In addition, tryptic peptides and chymotryptic subpeptides covering the whole extension of the light chain were obtained and characterized to further verify the sequence of the variable region and the established sequence of the constant region. The proposed sequence of the variable region indicates that it may be assigned to subgroup III. Positions 152 (serine) and 189 (arginine) correspond to the isotypic markers Kern- and Oz-, respectively. In addition, a novel substitution has been detected in the constant region where at position 155 isoleucine replaces the usually occurring valine.     

The primary structure of the Bence-Jones protein Kue. The amino acid sequence of the variable part of a human L-chain of the kappa-type (author's transl)]

The complete primary structure of the variable region of Bence-Jones protein Kue was elucitated with the aid of a few tryptic peptides, as well as one chymotryptic and one BNPS-scatol fragment. As a consequence of the evident homologies to other proteins this protein belongs to subgroup k/I. Protein Kue has some amino acid exchanges in certain positions in common with other proteins, probably giving rise to a new sub-subgroup. The constant region shows no amino acid exchanges in comparison with other human kappa L-chains. With valine covering position 191, protein Kue should be grouped per definition as an allotype Km (3).     

Comparative study on the structure of the light chains of human immunoglobulins. II. Assignment of a new subgroup.

The primary structure of the variable region of the human lambda type Bence Jones protein NIG-48 was determined by analysis of the N-terminal sequence of the completely reduced and aminoethylated protein, as well as of five cyanogen bromide fragments. The variable region of NIG-48 contains 112 amino acid residues. The protein NIG-48, having a unique sequence of the variable region, a low degree of homology (about 50%) with lambda chains of the five other subgroups and the addition of two residues around 65, may represent a new subgroup, namely V lambda VI.     

Protein L from Peptostreptococcus magnus binds to the kappa light chain variable domain.

Protein L is an immunoglobulin light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. The major variable region subgroups of human kappa and lambda light chains were tested for protein L binding; V kappa I, V kappa III, and V kappa IV bound protein L, whereas no binding occurred with proteins of the V kappa II subgroup or with any lambda light chain subgroups. Studies of the protein L binding capacity of naturally occurring VL fragments, and VL- and CL-related trypsin- and pepsin-derived peptides prepared from a kappa I light chain, localized the site of interaction to the VL domain. The affinity constant for the binding to an isolated V kappa I fragment was comparable to that for the native protein (Ka 0.9 x 10(9) M-1 and Ka 1.5 x 10(9) M-1, respectively). No binding occurred with CL-related fragments. Extensive reduction and alkylation of the V kappa fragment or the native kappa chain resulted in complete loss of protein L binding. Although it is possible, from comparative amino acid sequence data, to identify certain VL-framework region residues that account for the selective binding of protein L by kappa I, kappa III, and kappa IV proteins, our studies indicate that this interaction is essentially dependent upon the tertiary structural integrity of the kappa chain VL domain.     

Amino acid sequence of the light chain derived from a rabbit anti-p-azobenzoate antibody of restricted heterogeneity

The amino acid sequence of the light chain from a specifically purified rabbit (No. 2717) anti-p-azobenzoate antibody preparation (b4 allotype) of restricted heterogeneity has been determined. This light chain is composed of 216 residues, including seven half-cystine residues located at positions 23, 80, 88, 134, 171, 194 and 216. Three intrachain disulfide bonds appear to be present in contrast to only two disulfide bonds as has been so far described for Bence Jones protein and light chains of human and mouse. This light chain was sequenced by isolating the tryptic peptides, sequencing the peptides and establishing their order within the molecule. Unambiguous identification of the overlaps was achieved by taking into account the partially characterized tryptic peptides from citraconic anhydride-treated light chains and chymotryptic and peptic peptides from digests of both untreated and citraconylated light chains. Comparison of this amino acid sequence with the amino acid sequence of the car?ylterminal half of the b4 light chains from unimmunized rabbits reveals differences at positions 165, 166, 169 and 176 indicating the existence of more than one sequence in the b4 “constant” region. There is substantial sequence homology between the variable half of 2717 light chain and human Bence Jones protein. Indeed, 46 positions in the V region (42%) are occupied by the same residues in this light chain and in human subgroup V_κIII.     

Molecular cloning of a human immunoglobulin lambda chain variable sequence

We have cloned a human V lambda cDNA sequence from an Ig lambda-producing human Burkitt lymphoma cell line (BL2) by taking advantage of a cloned constant region gene as a primer for cDNA synthesis instead of an oligo(dT) primer. The amino acid sequence deduced from the nucleotide sequence of V lambda clones is highly related to that of the NEW V lambda protein of subgroup I. Southern blot hybridization of human DNAs with the V lambda I probe showed at least 12 hybridizing V lambda fragments. These fragments are amplified in K562 cells which derive from a case of chronic myelogenous leukemia and contain an amplified c-abl oncogene and amplified C lambda sequences.     

Is the formation of AL-type amyloid promoted by structural peculiarities of immunoglobulin L-chains? Primary structure of an amyloidogenic lambda-L-chain (BJP-ZIM)

A Bence-Jones protein (Protein ZIM) was isolated from the urine of a patient with myeloma-associated amyloidosis. The amino-acid sequence of the variable region of the carboxymethylated protein was established by automatic stepwise degradation of the enzymatically deblocked protein and tryptic peptides thereof. The protein is of the lambda-type of human immunoglobulin L-chains and is closely homologous to subgroup I. In the course of the tryptic digestion a precipitate was formed which showed properties characteristic of amyloid, such as staining with Congo red and green birefringence in polarized light. High-performance liquid chromatography was applied to separate these peptides. The precipitate consists of two peptides which coincide with position 19-45 of the variable and 129-140 of the constant part, respectively. Possible implications of this finding are discussed in the context of amyloid formation after limited proteolytic digestion.     

Structural studies of a carbohydrate-containing immunoglobulin-lambda-light-chain amyloid-fibril protein (AL) of variable subgroup III.

The amino acid sequence of the variable region of a carbohydrate-containing amyloid-fibril protein MOL of immunoglobulin-light-chain type (AL) was elucidated. The sequence determination involved cleaving the protein with CNBr, BNPS-skatole, thermolysin and trypsin. The sequenced protein consisted of about 130 amino acid residues; however, gel-filtration and N-terminal analysis studies revealed AL proteins ranging in Mr from about 10,000 to 25,000. The oligosaccharide chain was found to be bound in the hypervariable region. By sequence homology to other lambda chains the AL protein MOL was shown to be of the V lambda III subgroup.     

Molecular characterization of a human anti-Rh(D) antibody with a DH segment encoded by a germ-line sequence.

The lambda-light-chain and lambda-heavy-chain variable-region genes of an anti-Rh(D) (Rh, Rhesus; D, heavy-chain diversity region) human monoclonal antibody secreted by lymphocytes transformed by the Epstein-Barr virus have been cloned and sequenced. Sequence comparison of the anti-Rh(D)mAb lambda-chain variable region with those of the other available human lambda chains revealed that it belonged to the human V lambda I (V lambda, variable region of lambda chain) subgroup. The greatest sequence similarity (80%) was observed with that of another anti-Rh antibody lambda-chain directed against the Rh(c) antigen. For the VH (VH, variable region of heavy chain) sequence, the highest similarity (86%) was observed with the germline VHG3 gene which belongs to the VHI subgroup. The expressed DH sequence of the anti-Rh(D) antibody is also of germline origin and complementarity-determining region 3 is thus produced by VH-DH and DH-JH (J, joining region) joining without recombination of multiple DH gene segments.     

Cloning and sequence analysis of an Ig lambda light chain mRNA expressed in the Burkitt''s lymphoma cell line EB4.

A cDNA library was constructed from the mRNA of the Ig lambda producing Burkitt's lymphoma cell line, EB4. Overlapping clones encompassing the coding sequence of the Ig lambda mRNA were isolated and sequenced. The predicted amino acid sequence shows a short hydrophobic leader peptide and a mature polypeptide of 217 residues in which V, J and C regions can be distinguished. The V region belongs to subgroup VI and has greatest homology (80%) with the Amyloid-AR protein. The constant region is the Kern- Oz+ isotype. Probing normal human DNA with the subcloned V lambda coding sequence detects one gene at high stringency and a family of 11 members at low stringency. To date, no restriction enzyme site polymorphisms have been detected. The V lambda VI gene is rearranged on both chromosomes of EB4 and is deleted on both chromosomes in the Burkitt's lymphoma cell line BL2.     

The primary structure of the constant region of Basilea-rabbit immunoglobulin lambda-chains.

The amino acid sequence of the constant region of the immunoglobulin lambda-chain of the rabbit (C lambda) has been determined. This chain was obtained from a homogeneous anti-(type II pneumococcal polysaccharide) antibody fraction raised in Basilea rabbit 4548. The C lambda-region sequence was established by the analysis of tryptic and some overlapping chymotryptic peptides and partly by homology with known C lambda-region sequences of lambda-chains from other species. Peptides were isolated by a combination of methods including high-voltage paper electrophoresis, gel filtration and high-pressure liquid chromatography. The peptides were subjected to automated Edman degradation in the presence of Polybrene. The sequence showed no evidence of heterogeneity. Large interspecies similarities, as well as amino acid interchanges, are apparent among various C lambda regions; the degrees of homology between rabbit C lambda region and the C lambda region from human, porcine and murine chains are 72.6, 71 and 72% respectively. The similarity of lambda-chains of different species is much greater than that found between rabbit kappa- and lambda-chains (about 34% homology). Genetic implications of these results are discussed.     

Primary structure of a human IgA1 immunoglobulin. IV. Streptococcal IgA1 protease, digestion, Fab and Fc fragments, and the complete amino acid sequence of the alpha 1 heavy chain.

In order to establish the complete amino acid sequence of the human IgA alpha1 chain Bur, IgA1 protease from Streptococcus sanguis was employed to generate Fabalpha and Fcalpha fragments in the final stage of this investigation. Cyanogen bromide cleavage of the Fabalpha fragment followed by reduction and aminoethylation produced the Fd' fragment (residues 84 to 227); this contains part of the variable region (VR), the whole first constant domain (Calpha1), and part of the hinge region of this heavy chain. The tryptic peptides of the Fd' fragment were isolated, characterized, and sequenced. The results together with the data in the preceding papers on chymotryptic, tryptic, and thermolysin peptides permitted the complete amino acid sequence of the human IgA alpha1 chain to be established.     

The amino acid sequence of a carbohydrate-containing immunoglobulin-light-chain-type amyloid-fibril protein.

The amino acid sequence of an amyloid-fibril protein Es492 of immunoglobulin-lambda-light-chain origin (AL) was elucidated. The amyloid fibrils were obtained from the spleen of a patient who died from systemic amyloidosis. The amino acid sequence was elucidated from structural studies of peptides derived from digestion of the protein with trypsin, thermolysin, chymotrypsin and Staphylococcus aureus V8 proteinase and from cleavage of the protein with CNBr and BNPS-skatole. A heterogeneity in the length of the polypeptide was seen in the C-terminal region. The protein was by sequence homology to other lambda-chains shown to be of the V lambda II subgroup. Although an extensive homology was seen, some amino acid residues in positions 26, 31, 32, 40, 44, 93, 97, 98 and 99 have not previously been reported in these positions of V lambda II proteins. The significance of these residues in the fibril formation is unclear. The protein was found to contain carbohydrate, with glycosylation sites in two of the hypervariable regions.     

Anti-lambda-light chain-peptide antibodies are suitable for the immunohistochemical classification of AL amyloid

We aimed to test whether antibodies raised against recombinant peptides corresponding to the variable region of immunoglobulin light chains are suitable for the immunohistochemical classification of amyloid. The Entrez database of the National Center for Biotechnology Information (NCBI) was searched for all protein sequence entries which met the search criteria "amyloid" and "lambda light chain". Sixty-four different lambda-light chain-derived amyloid protein sequences were retrieved, aligned and categorized into the V region subgroups of lambda-light chain detailed by the NCBI, i.e. subgroup I (21 protein sequences), II (14), III (6), IV (1), V (1) and VI (21). V region subgroup I was chosen for epitope sequence selection and two rabbits were immunized with the following peptides: NH2-ISCSGSSSNIGSNTV-CONH2 and NH2-QRPSG VPDRFSGSKSGTS-CONH2. Sensitivity and specificity of the IgG-purified antibodies was tested by Western blotting using amyloid A- (AA), ALlambda- and ALkappa-amyloid proteins, and by immunohistochemistry on tissue microarrays with 110 different amyloid containing tissue samples obtained at autopsy from 22 patients, and on 27 biopsy specimens from a series of 24 patients. Our peptide antibodies specifically stained AL amyloid lambda-light chain-origin, in both Western blots and formalin-fixed and paraffin-embedded tissue sections, confirming that peptide-antibodies directed against immunoglobulin-derived lambda-light chain proteins can be applied for the immunohistochemical classification of amyloid. This offers the opportunity to generate a large set of anti-lambda-light chain protein-antibodies for the immunohistochemical classification of amyloid independently from native human tissue sources.     

Cloning and nucleotide sequence analysis of the cDNA of the immunoglobulin lambda light chain from the bovine udder

A cDNA library was constructed from the mRNA of bovine mammary gland which contained Ig lambda producing plasmacytes. Overlapping clones encompassing the major portion of the coding sequence of the Ig lambda mRNA were isolated and sequenced. Predicted amino acid sequence shows a mature polypeptide of 217 residues in which V, J and C regions can be distinguished. The constant region has greatest homology with human Ig lambda mRNA constant region. The area of the V region between CDR3 and CDR2 has also great homology with the same area of human lambda chain.     

Rule of antibody structure. Primary structure of a human monoclonal IgAl-immunoglobulin (myeloma protein Tro). VI. Amino acid sequence of the L-chain, lambda-type, subgroup II]

The primary structure of the L-chain of an IgA1-immunoglobulin (Myeloma protein Tro) has been determined by means of cleavage with trypsin and, if necessary, with alpha-chymotrypsin. The tryptic peptides of the variable part were characterized by amino acid analysis, Dansyl-Edman degradation and cleavage with carboxypeptidase; the peptides of the constant part were identified by amino acid analyses and determination of its N- and C-terminal residues. The sequence of the remaining amino acids and the arrangement of the peptides were established in homology to known structures. The protein comprises 216 amino acids. The homology of the variable part clearly characterizes it as belonging to subgroup II of lambda-chains. In positions 27a, b and c, there are the subgroup-specific additional residues and in position 96 is the characteristic deletion. The constant part of the chain is Kern- and Oz- which indicates that it has serine in position 154 and arginine in position 191.     

Isolation and sequence determination of cDNA clones for porcine and human lipoamide dehydrogenase. Homology to other disulfide oxidoreductases

A 2.3-kilobase cDNA clone encoding lipoamide dehydrogenase was isolated from a porcine adrenal medulla library in the vector pCD by screening with four synthetic oligonucleotide probes corresponding to amino acid sequence from tryptic peptides of porcine lipoamide dehydrogenase. A 450-bp fragment of the porcine cDNA was used to screen a human small cell lambda gt10 library at reduced stringency. Overlapping human cDNA clones of various lengths were isolated, the largest of which was again 2.3 kilobases in length. Sequencing of both porcine and human cDNAs revealed a short 5'-untranslated region followed by 1530-bp of coding region and 700 bp of 3'-untranslated region preceding a poly(A) tail. The porcine cDNA displayed coding regions corresponding to the known tryptic peptides and a 35-amino acid leader sequence involved in targeting of the protein to the mitochondria. The human lipoamide dehydrogenase cDNA is 96% identical to the porcine at the amino acid level. Alignment of the deduced amino acid sequence of human lipoamide dehydrogenase with human erythrocyte glutathione reductase and mercuric reductase from Tn501 revealed extensive homologies throughout the primary sequence, suggesting that secondary and tertiary structure is also similar among these three enzymes.     

Amyloid fibrils derived from V-region together with C-region fragments from a lambda II-immunoglobulin light chain (HAR)

Amyloid fibril proteins were isolated from the spleen of a patient with IgD(lambda)-plasmocytoma by extraction and gel filtration in 5M guanidine hydrochloride. The molecular mass of the predominant polypeptide chain was approximately 5000 Da. Its complete amino-acid sequence was elucidated by stepwise automated degradation of the carboxymethylated polypeptide chain and by structural studies of tryptic and thermolysinolytic cleavage products. The length of the polypeptide chain was 58 to 59 residues and it was homologous to the amino acids in positions 8 through 65 of the variable part of an lambda-type immunoglobulin light chain, which was most closely related to the lambda II subgroup. The N-terminal sequence of this amyloid fibril protein proved to be heterogeneous, indicating cleavage after the amino acids in positions 7 and 8. Peptides from the constant part of the lambda-chain were unexpectedly found in the tryptic digest of the denatured amyloid protein HAR. One polypeptide derived from the constant region was separated from the main component by high performance liquid chromatography. Its amino-acid sequence commenced at position 111 and could be traced in 41 steps. In this case, at least two constant region fragments were shown to be constituents of the amyloid fibril protein. The association of fragments from the variable as well as the constant region is discussed with respect to amyloid formation.     

Primary structure of the variable region of a human lambda VI light chain: Bence Jones protein SUT

To ascertain if lambda VI light chains have unique structural features that account for the preferential association of these proteins with primary or multiple myeloma-related amyloidosis (amyloidosis AL) we have determined the complete amino acid sequence of the variable (V) region of the lambda VI Bence Jones protein SUT. This protein, obtained from a patient with amyloidosis AL, represents a complete light chain consisting of 216 residues and it has structural and serologic properties characteristic for lambda VI light chains. The sequence of the joining segment (J) (positions 100 to 111) of protein SUT is identical to that of the J lambda I segment of the mouse IG lambda light chain gene. V region SUT is closely homologous in sequence to that of another lambda VI amyloid fibrillar protein, AR, differing by 21 residues. The V regions of proteins SUT and AR contain a two-residue insertion at positions 68 and 69 that has also been found in two other lambda VI human light chains but not in the lambda-chains of other V region subgroups.