Catalytic digestion of human tumor necrosis factor-α by antibody heavy chain.
It has long been an important task to prepare a catalytic antibody capable of digesting a targeting crucial protein that controls specific life functions. Tumor necrosis factor-α (TNF-α) is a cytokine and an important molecule concerned with autoimmune diseases such as rheumatoid arthritis, chronic obstructive pulmonary disease, and Crohn's disease. A mAb (ETNF-6 mAb) raised against human TNF-α was prepared, and the steric conformation was created by using molecular modeling after the cDNA was sequenced. The heavy chain (ETNF-6-H) of the mAb was considered to possess a catalytic triad-like structure in the complementarity determining regions (CDRs). As a result, ETNF-6-H exhibited a peptidase and a protease activity. In fact, ETNF-6-H predominantly cleaved the Ser5-Arg6 bond of TNF-α at the first step, resulting in the generation of a fragment of ∼ 17 kDa. This fragment was digested to a smaller molecule of 15 kDa by scission of the Gln21-Ala22 bond. The intermediate product was further converted into a fragment of 13.3 kDa by successive cleavage of the Leu36-Leu37 and Asn39-Gly40 bonds. The heavy chain possessed a protease activity against TNF-α with a multicleavage site.
Hifumi
Emi
E
Research Center for Applied Medical Engineering, Oita University, Oita, Japan.
Higashi
Kyohei
K
Uda
Taizo
T
D000595:Amino Acid Sequence; D000818:Animals; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D000915:Antibody Affinity; D001483:Base Sequence; D055162:Biocatalysis; D003429:Cross Reactions; D005260:Female; D006801:Humans; D006868:Hydrolysis; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology* / Q000378:metabolism*
D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha