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

Journal Article · · Journal of Biological Chemistry
 [1];  [2];  [2];  [2];  [2];  [2];  [2];  [3];  [4];  [2]
  1. National Autonomous Univ. of Mexico, Mexico City (Mexico). Dept. of Molecular Medicine and Bioprocesses; DOE Office of Scientific and Technical Information (OSTI)
  2. National Autonomous Univ. of Mexico, Mexico City (Mexico). Dept. of Molecular Medicine and Bioprocesses
  3. National Autonomous Univ. of Mexico, Mexico City (Mexico). Dept. of Molecular Biology of Plants. Inst. of Biotechnology
  4. National Autonomous Univ. of Mexico, Mexico City (Mexico). Lab. of Physical Chemistry and Protein Engineering. Faculty of Medicine
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Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this paper, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. Finally, this mutagenic approach helped to identify key regions implicated in λ3 AL.

Research Organization:
National Autonomous Univ. of Mexico, Mexico City (Mexico)
Sponsoring Organization:
USDOE; National Inst. of Health (NIH) (United States); National Council of Science and Technology (CONACYT) (Mexico)
Grant/Contract Number:
AC02-98CH10886
OSTI ID:
1349050
Journal Information:
Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 5 Vol. 290; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
English

References (52)

Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four Vλ6 proteins journal June 2004
Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light-chain amyloid proteins journal March 1995
Immunoglobulin kappa light chain and its amyloidogenic mutants: A molecular dynamics study: Amyloidogenic Mutants of IG Light Chain journal February 2004
Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains journal February 2008
Extended Analysis of AL-Amyloid Protein from Abdominal Wall Subcutaneous Fat Biopsy: Kappa IV Immunoglobulin Light Chain journal April 1998
Sequence and Evolution of the Human Germline VλRepertoire journal November 1996
Review: Immunoglobulin Light Chain Amyloidosis—The Archetype of Structural and Pathogenic Variability journal June 2000
Simultaneous Optical Recording of Membrane Potential and Intracellular Calcium from Brain Slices journal June 1999
Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavine T journal March 1989
By-passing immunization: Human antibodies from V-gene libraries displayed on phage journal December 1991
Protein misfolding and disease; protein refolding and therapy journal June 2001
The protein data bank: A computer-based archival file for macromolecular structures journal May 1977
An alternative approach in the structure-based predictions of the thermodynamics of protein unfolding journal November 1997
Clinical aspects of systemic amyloid diseases journal November 2005
Inference of Macromolecular Assemblies from Crystalline State journal September 2007
Thermodynamic and Kinetic Characterization of a Germ Line Human λ6 Light-Chain Protein: The Relation between Unfolding and Fibrillogenesis journal March 2009
Structural Alterations within Native Amyloidogenic Immunoglobulin Light Chains journal May 2009
A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring λ6 Light-Chain Fibrillogenesis journal February 2010
The carboxy terminus of the .beta. amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer's disease journal May 1993
Amino acid sequence of the variable region of the light (λ) chain from human myeloma cryoimmunoglobulin IgG Hil journal May 1978
Structural Characterization of the Partially Folded Intermediates of an Immunoglobulin Light Chain Leading to Amyloid Fibrillation and Amorphous Aggregation † journal March 2007
Thermodynamic Instability of Human λ6 Light Chains:  Correlation with Fibrillogenicity † journal October 1999
Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins journal September 2004
Therapeutic strategies for human amyloid diseases journal April 2002
Differences in kappa to lambda (κ:λ) ratios of serum and urinary free light chains journal December 2001
Natural  -sheet proteins use negative design to avoid edge-to-edge aggregation journal March 2002
A role for destabilizing amino acid replacements in light-chain amyloidosis. journal June 1994
Altered Dimer Interface Decreases Stability in an Amyloidogenic Protein journal April 2008
Structural Insights into the Role of Mutations in Amyloidogenesis journal September 2008
Biochemical and aggregation analysis of Bence Jones proteins from different light chain diseases journal January 2008
PIC: Protein Interactions Calculator journal May 2007
Recursive PCR: a novel technique for total gene synthesis journal January 1992
Phaser crystallographic software journal July 2007
PROCHECK: a program to check the stereochemical quality of protein structures journal April 1993
Coot model-building tools for molecular graphics journal November 2004
Scaling and assessment of data quality journal December 2005
The integration of macromolecular diffraction data journal December 2005
ALINE : a WYSIWYG protein-sequence alignment editor for publication-quality alignments journal April 2009
XDS journal January 2010
PHENIX: a comprehensive Python-based system for macromolecular structure solution journal January 2010
Pitfalls of Molecular Replacement: the Structure Determination of an Immunoglobulin Light-Chain Dimer journal November 1996
Refinement of Macromolecular Structures by the Maximum-Likelihood Method journal May 1997
Protein Misfolding, Functional Amyloid, and Human Disease journal June 2006
Analysis of Vλ-Jλ expression in plasma cells from primary (AL) amyloidosis and normal bone marrow identifies 3r(λIII) as a new amyloid-associated germline gene segment journal August 2002
Immunoglobulin light chain variable (V) region genes influence clinical presentation and outcome in light chain–associated amyloidosis (AL) journal May 2003
Dangerous small B-cell clones journal October 2006
The tropism of organ involvement in primary systemic amyloidosis: contributions of Ig VL germ line gene use and clonal plasma cell burden journal August 2001
AGGRESCAN: a server for the prediction and evaluation of "hot spots" of aggregation in polypeptides journal January 2007
Insight into the Structure of Amyloid Fibrils from the Analysis of Globular Proteins journal January 2006
Mutations in Specific Structural Regions of Immunoglobulin Light Chains Are Associated with Free Light Chain Levels in Patients with AL Amyloidosis journal April 2009
Primary (AL) Amyloidosis in Plasma Cell Disorders journal July 2006
Towards Understanding the Structure-Function Relationship of Human Amyloid Disease journal February 2004

Cited By (3)

Concurrent structural and biophysical traits link with immunoglobulin light chains amyloid propensity journal December 2017
Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains journal January 2017
Different Dynamics in 6aJL2 Proteins Associated with AL Amyloidosis, a Conformational Disease journal August 2019

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
Amyloid
Antibody
Fibril
Mutagenesis in Vitro
Thermodynamics