RESUMO
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'.
Assuntos
Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina M/química , Imunoglobulina M/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Proteína de Bence Jones/química , Proteína de Bence Jones/genética , Cristalografia por Raios X , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Imunoglobulinas/química , Imunoglobulinas/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Mieloma/química , Proteínas do Mieloma/genética , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Eletricidade Estática , Macroglobulinemia de Waldenstrom/genética , Macroglobulinemia de Waldenstrom/imunologiaRESUMO
The X-ray structure of an immunoglobulin light-chain dimer isolated from the urine as a "Bence-Jones protein" from a patient with multiple myeloma and amyloidosis (Sea) was determined at 1.94 A resolution and refined to R and R(free) factors of 0.22 and 0.25, respectively. This "amyloidogenic" protein crystallized in the orthorhombic P2(1)2(1)2(1) space group with unit-cell parameters a = 48.28, b = 83.32, c = 112.59 A as determined at 100 K. In the vital organs (heart and kidneys), the equivalent of the urinary protein produced fibrillar amyloid deposits which were fatal to the patient. Compared with the amyloidogenic Mcg light-chain dimer, the Sea protein was highly soluble in aqueous solutions and only crystallized at concentrations approaching 100 mg ml(-1). Both the Sea and Mcg proteins packed into crystals in highly ordered arrangements typical of strongly diffracting crystals of immunoglobulin fragments. Overall similarities and significant differences in the three-dimensional structures and crystalline properties are discussed for the Sea and Mcg Bence-Jones proteins, which together provide a generalized model of abnormalities present in lambda chains, facilitating a better understanding of amyloidosis of light-chain origin (AL).