Functional characterization of 3 thioredoxin homology domains of ERp72.
Folding of secretory proteins is associated with the formation and isomerization of disulfide bonds. ERp72, a protein disulfide isomerase (PDI) family member, possesses 3 thioredoxin homology domains, but the participation of each domain in disulfide-bond formation and isomerization remains to be determined. We analyzed the function of individual domains in the insulin reduction assay system by site-directed mutagenesis with cysteine-to-serine replacement. All domains contributed to apparent steady-state binding (Km) and catalysis at saturating substrate concentrations (kcat) but in different manners. A mutant ERp72 with mutations in domains 1 and 2 (ERp72-mut-1+2) exhibited reductions in kcat of 73.9% when compared with wild type, whereas ERp72-mut-1+3 (mutations in domains 1 and 3) and ERp72-mut-2+3 (mutations in domains 2 and 3) exhibited less substantial reductions in kcat. ERp72-mut-1+3 and ERp72-mut-2+3 showed elevations in Km of 89.9% and 96.2%, respectively, when compared with wild type, whereas ERp72-mut-1+2 exhibited smaller elevations in Km. These results suggest that domains 1 and 2 make greater contributions to catalyzing efficacy and domain 3 to binding affinity. Domain 2 is involved in binding affinity, in combination with domain 3, in addition to its own contribution to catalyzing efficacy. This assignment of functions to individual domains is similar to that observed in other PDI domains, which is consistent with the high sequence homology between ERp and PDI domains.
Satoh
Mamoru
M
Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan.
Shimada
Atsuyoshi
A
Keino
Hiromi
H
Kashiwai
Akiko
A
Nagai
Naoko
N
Saga
Shinsuke
S
Hosokawa
Masanori
M
D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides; D007328:Insulin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D013879:Thioredoxins / Q000235:genetics*
D004220:Disulfides; D007328:Insulin; D008562:Membrane Glycoproteins; C062477:endoplasmic reticulum glycoprotein p72; D013879:Thioredoxins; D019704:Protein Disulfide-Isomerases