Deborah T. Weiss

Transgenic Mouse Model of AA Amyloidosis

AA amyloidosis can be induced in mice experimentally through injection of certain chemical or biological compounds. However, the usefulness of this approach is limited by its dependence on exogenous inflammatory agents that stimulate cytokines to increase the synthesis of precursor serum amyloid A (SAA) protein and the transitory nature of the pathological fibrillar deposits. We now report that transgenic mice carrying the human interleukin 6 gene under the control of the metallothionein-I promoter had markedly increased concentrations of SAA and developed amyloid in the spleen, liver, and kidneys by 3 months of age. At the time of death about 6 months later, organs obtained from these animals had extensive amyloid deposits. This disease process was apparent radiographically using small-animal computer axial tomography and magnetic resonance imaging equipment. The AA nature of the amyloid was evidenced immunohistochemically and was unequivocally established by sequence analysis of protein extracted from the fibrils. The availability of this unique in vivo experimental model of AA amyloidosis provides the means to assess the therapeutic efficacy of agents designed to reduce or prevent the fibrillar deposits found in AA and other types of amyloid-associated disease.

Protein and host factors implicated in the pathogenesis of light chain amyloidosis (AL amyloidosis)

(1995). Protein and host factors implicated in the pathogenesis of light chain amyloidosis (AL amyloidosis) Amyloid: Vol. 2, No. 4, pp. 269-279.

Differences in kappa to lambda (κ:λ) ratios of serum and urinary free light chains

Free light chains (FLC) are a natural product of B lymphocytes and, as such, represent a quantifiable biomarker of cellular proliferation. Accurate measurement of the concentrations of these components in serum and urine provides a unique means of ascertaining B cell immunoglobulin synthesis during physiologic and, especially, pathologic states, where such information has important diagnostic and therapeutic implications. Previously, use of such quantitative assays has been limited due to the lack of potent serologic reagents specific for these components. We have immunized mice with κ‐ and λ‐type monoclonal human light chains (Bence Jones proteins (BJP)) and have obtained monoclonal antibodies (MoAbs) that differentiate between unbound and bound light chains. These highly specific MoAbs were used to measure by ELISA the concentrations of FLC in the serum of 22 normal individuals and in urine from 16 of these subjects. The mean serum κ and λ FLC concentrations were found to be 16.6u2003±u20036.1u2003μg/ml and 33.8u2003±u200314.8u2003μg/ml, respectively. In contrast, the values for urinary κ and λ FLC were 2.96u2003±u20031.84u2003μg/ml and 1.07u2003±u20030.69u2003μg/ml, respectively. In each case studied, the serum κ:λ ratio was consistently less than that of urine (mean values, serum ≈u20031:2; urine ≈u20033:1). That the rate of synthesis of λ‐type FLC exceeded that of κ was evidenced in assays of culture fluid supernatants of unstimulated normal peripheral blood mononuclear cells (PBMC), where the mean κ:λ ratio was determined to be 1:1.4. Metabolic studies in which mice were injected with pools of κ‐ and λ‐type BJP prepared in ratios of 1:1, 1:2 and 1:4 demonstrated that, regardless of the proportion, κ FLC were preferentially excreted. Our studies provide the first evidence that λ FLC are secreted by normal PBMC at a greater rate than are κ FLC, as evidenced in biosynthetic studies and by measurement of their serum concentrations. Further, we posit that quaternary structural differences between the two light‐chain isotypes may account for the predominance of κversusλ components in urine.

Tertiary structure of human lambda 6 light chains.

AL amyloidosis is a disease process characterized by the pathologic deposition of monoclonal light chains in tissue. To date, only limited information has been obtained on the molecular features that render such light chains amyloidogenic. Although protein products of the major human V kappa and V lambda gene families have been identified in AL deposits, one particular subgroup--lambda 6--has been found to be preferentially associated with this disease. Notably, the variable region of lambda 6 proteins (V lambda 6) has distinctive primary structural features including the presence in the third framework region (FR3) of two additional amino acid residues that distinguish members of this subgroup from other types of light chains. However, the structural consequences of these alterations have not been elucidated. To determine if lambda 6 proteins possess unique tertiary structural features, as compared to light chains of other V lambda subgroups, we have obtained x-ray diffraction data on crystals prepared from two recombinant V lambda 6 molecules. These components, isolated from a bacterial expression system, were generated from lambda 6-related cDNAs cloned from bone marrow-derived plasma cells from a patient (Wil) who had documented AL amyloidosis and another (Jto) with multiple myeloma and tubular cast nephropathy, but no evident fibrillar deposits. The x-ray crystallographic analyses revealed that the two-residue insertion located between positions 68 and 69 (not between 66 and 67 as previously surmised) extended an existing loop region that effectively increased the surface area adjacent to the first complementarity determining region (CDR1). Further, an unusual interaction between the Arg 25 and Phe 2 residues commonly found in lambda 6 molecules was noted. However, the structures of V lambda 6 Wil and Jto also differed from each other, as evidenced by the presence in the latter of certain ionic and hydrophobic interactions that we posit increased protein stability and thus prevented amyloid formation.

Molecular characterization of a human VλVIII germline gene

Abstract Human λ light chains of the recently recognized variable region (V L ) subgroup V λVIII can be distinguished from proteins of other V λ gene families on the basis of distinctive chemical and serologic properties and by their preferential association with certain types of autoantibodies, i.e. rheumatoid factors (RFs). We now report that we have cloned from a human placental library a V λVIII -encoding germline gene, designated IGLV8A1, using as a molecular probe a partial V λVIII fragment generated by polymerase chain reaction (PCR) from genomic DNA. IGLV8A1 contained all the requisite elements of a potentially functional gene, including a V λ exon with an open reading frame encoding 103 residues. Its expressed products were identified through analyses of cDNA cloned from two different monoclonal λVIII B-cell populations. The primary structure of λVIII light chains differed from that of λI, λII, λIII, λIV and λVI proteins by the presence of distinctive residues within the first framework region (FR1) and an 11- rather than 7-residue second complementarity-determining region (CDR2). Remarkably, the IGLV8A1 gene was more homologous to the two functional rabbit V λ germline genes, RVλ2 and RVλ3 (including the presence of one extra codon within the leader sequence), and to the murine V λx gene. Light chains encoded by the human, rabbit and mouse λVIII-related genes shared certain unique primary structural features, notably the four additional CDR2 residues. The evolutionary conserved nature of the human V λ gene and, in particular, the apparently novel tertiary structural effects induced by an elongated CDR2 provide evidence for the biological and functional importance of the V λVIII subgroup.

Identification and characterization of a human Vλ5 (T1) germline gene that encodes structurally unique λ light chains

Abstract The human germline V λ repertoire consists of about 30 functional genes that have been classified into 10 families on the basis of homologies in nucleotide sequences that encode approximately the first 96 to 104 residues of λ light chains. One family, termed V λ 5, is of special interest because the λ light chain products of these genes have unique structural features. We have now isolated from genomic DNA one member of this family, designated IGLV5-1, using as a molecular probe a partial V λ 5-germline-gene fragment generated by polymerase chain reaction. IGLV5-I contains all the requisite elements of a potentially functional gene, including a V λ exon with an open reading frame specifying 104 residues. A V λ 5-related cDNA (ZW) was also cloned from a bone marrow-derived plasma-cell population obtained from a patient with light-chain-associated (AL) amyloidosis. Comparison of the predicted protein sequences encoded by the IGLV5-I-germline gene. cDNA ZW, and three other reported V λ 5-related cDNAs with those of the deduced or expressed products of the other nine known human V λ -gene families revealed that V λ 5 proteins contain distinctive primary structural features. These include the presence within the second complementarity determining region (CDR2) and the third framework region (FR3) of 11 and 34 amino acids. respectively, rather than the 7 and 32 that occur in the most commonly expressed V λ 1-, V λ 2- and V λ 3-type light chains. Although certain of the V λ -gene families encode either an elongated CDR2 or FR3. V λ 5 proteins are remarkable in that they have additional residues in both regions of the molecule. In this respect, these polypeptides are most similar to surrogate light-chain-associated human and mouse VpreB components that also have these unusual primary structural features. Further, the four additional CDR2 residues and the two-residue FR3 insertion have been found among λ-type light chains of certain non-mammalian species. The evolutionarily conserved nature of human V λ 5-related genes and, in particular, the presumably novel tertiary structural effects induced by the unique features of the λ light chains encoded by these elements suggest that the V λ 5-gene family has biological and functional importance.

Production of Amyloidogenic Peptides from Human Immunoglobulin Light (L)-Chains

Structural studies were performed on three human immunoglobulin L-chains isolated from the urine of patients with generalized amyloidosis. The complete amino acid sequence of the V-regions was established for all three proteins. According to their amino acid sequence all proteins belong to Subgroup I of human k-L-chains. Heavy precipitates were formed during the tryptic digestion of the reduced and carboxymethylated chains. These precipitates resemble amyloid in many ways as they stained positive with Congo red, showed the typical green birefringence in polarized light, were insoluble in most solvents and indigestible by the usual proteolytic enzymes. Amylo-idlike fibrillar structures were demonstrable by electron microscopy in the precipitates of all three proteins. These amyloid forming peptides, which aggregate in the precipitate derive in all three chains from the same regions within the variable and constant parts in the three L-chains. Generally there is some evidence that the capability for amyloid formation resides in certain hydrophobic regions within the human immunoglobulin L-chains.

Equine light-chain-associated amyloidosis

We have previously immunologically typed amyloid protein extracted from a horse with malignant histiolymtphocytic lymphosarcoma as immunoglobulin (Ig)-derived amylaid. In the present paper, the Ig character of the horse amyloid is confirmed by the amino acid sequence of the constituent protein. Its major component is identified as a 217-residue complete horse λ light chain. On the basis of sequence homology, the variable (V) and joining (J) segments were most closely related to those encoded by the equine Vλ2- and JλI-gene families, respectively. Notably, the constant (C) region, although most closely related to the CλI-gene, differed from the prototypic sequence by five residues. These findings have provided further evidence that proteolytic precleavage of the precursor is not essential in light-chain-associated (AL) amyloidogenesis. In addition to establishing the first complete amino acid sequence of a horse Ig light chain, the discovery of a unique C-region primary structure implies extensive CλI poly...

Experimental Production of Human Amyloidosis AL

Amyloidosis AL is characterized by the deposition in tissue of monoclonal light chains or light-chain fragments. The lack of an in vivo model has limited acquisition of information regarding the pathogenesis of this disease. We now report that it has been possible, for the first time, to induce human light-chain-related amyloidosis through use of an experimental mouse model. The injection into mice of Bence Jones proteins obtained from patients with amyloidosis AL resulted in the deposition of the human proteins as amyloid. The deposits exhibited Congo red positivity, birefringence, and a fibrillar ultrastructure. They were located primarily within the blood vessel walls of the mouse kidneys but were also found in spleen, liver, and other organs. In control experiments, mice injected in identical fashion with non-amyloid-associated Bence Jones proteins did not develop amyloidosis. The experimental mouse model provides a means to study further protein and host factors responsible for the development of human amyloidosis AL.