Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light‐chain amyloid proteins

Abstract

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VKIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VKIV products have tertiary structural features comparable to those of the patient-derived proteins. This welldefined set of three clinically characterized human KIV light chains, together with the capability to produce these KIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.Keywords: amyloid proteins; immunoglobulin light chains; kappa IV domains; recombinant human VL; synthetic genes; variable-domain dimerization Over the past 30 years, detailed analyses of the structure and biomutagenesis to investigate effects of the changes on biological physical properties of immunoglobulin molecules have probed activities; synthetic Ig genes have been generated for the promany aspects of Ig interactions and effector functions (Padlan, duction of unique antibody reagents for medical and diagnos-1994). More recently, Ig genes have been cloned and altered by tic purposes (Borrebaeck, 1992 its include the assembly of a primarily p-structure protein intoThe first two authors contributed equally to this project.nonbranching, insoluble fibrils of diameter 7-10 nm and a char-IPTG, isopropyl P-D-thiogalactopyranoside; TES, Tris-EDTA-sucrose lead to death' Defining features Of amy1oid depos-42 1

Structural Insights into the Role of Mutations in Amyloidogenesis

“…Crystallization studies of other AL proteins, AL-12 and AL-103, have also shown the 40 -44 loop to be disordered. 3 Therefore, this region may be a key to amyloidogenic propensity in LC proteins.…”

“…Although other precursor proteins may be wild type or linked to a single hereditary mutation, AL is distinct in that hypervariability yields a different set of mutations in each patient. Variable domains of LCs undergo somatic hypermutation, and in the case of AL patients (2), these mutations make proteins thermodynamically destabilized compared with non-amyloidogenic proteins (3)(4)(5). Some studies have linked the destabilizing somatic mutations present in AL proteins and the propensity to form amyloid fibrils that leads to cellular and organ damage (4,6,7).…”

Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains

“…Residue Pro-40 was of particular interest because it is highly conserved in both and isotypes. Furthermore, residue Pro-40 of the multiple myeloma LEN protein has been shown to confer stability onto several amyloid-related light chains (14). We evaluated the role of Pro-40 using the 3rJL2/YA/P40S mutant (3rJL2/C34Y/ W91A/P40S).…”

“…The comparison of amyloidogenic and nonamyloidogenic V L sequences has allowed other researchers to identify certain amino acid changes that destabilize the V L domain (12)(13)(14)(15)(16). The introduction of some of these mutations into nonamyloidogenic V L domains decreases their stability and increases their tendency to aggregate and form amyloid fibers in vivo (17).…”

“…Taking the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions on the stability and aggregation propensity of 3 light chains. Mutations were introduced into two regions thought to protect against light chain fibril formation: the sheet switch (positions 7 and 8) (13) and the loop 40 -60 region (positions 40 and 48) (14). X-ray crystallography revealed that the three-dimensional topology of the single and double 3r mutants was not significantly different.…”