Characterization of disulfide bonds in recombinant proteins: reduced human interleukin 2 in inclusion bodies and its oxidative refolding

Section: Tm L-[mentioning

confidence: 99%

Carbohydrate Recognition Site of Interleukin-2 in Relation to Cell Proliferation

Fukushima

Hara-Kuge

Ideo

et al. 2001

“…After incubation for 2.5 h at 37°C, IL-2 production was induced by addition of 0.5 mM isopropyl ␤-thiogalactoside, and the cells were grown for 2.5 h. IL-2 was produced mainly in inclusion bodies. The inclusion bodies were solubilized and IL-2 was refolded by a method described previously (18), with slight modification, as follows. The cells were collected by centrifugation and homogenized by lysozyme treatment and sonication at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Interleukin-2 Carbohydrate Recognition Modulates CTLL-2 Cell Proliferation

Fukushima

Yamashita

2001

Interleukin-2 (IL-2) specifically recognizes high-mannose type glycans with five or six mannosyl residues. To determine whether the carbohydrate recognition activity of IL-2 contributes to its physiological activity, the inhibitory effects of high-mannose type glycans on IL-2-dependent CTLL-2 cell proliferation were investigated. Man 5 GlcNAc 2 Asn added to CTLL-2 cell cultures inhibited not only phosphorylation of tyrosine kinases but also IL-2-dependent cell proliferation. We found that a complex of IL-2, IL-2 receptor ␣, ␤, ␥ subunits, and tyrosine kinases was formed in rhIL-2-stimulated CTLL-2 cells. Among the components of this complex, only the IL-2 receptor ␣ subunit was stained with Galanthus nivalis agglutinin which specifically recognizes high-mannose type glycans. This staining was diminished after digestion of the glycans with endo-␤-N-acetylglucosaminidase H or D, suggesting that at least a N-glycan containing Man 5 GlcNAc 2 is linked to the extracellular portion of the IL-2 receptor ␣ subunit. Our findings indicate that IL-2 binds the IL-2 receptor ␣ subunit through Man 5 GlcNAc 2 and a specific peptide sequence on the surface of CTLL-2 cells. When IL-2 binds to the IL-2R␣ subunit, this may trigger formation of the high affinity complex of IL-2-IL-2R␣, -␤, and -␥ subunits, leading to cellular signaling.

“…To separate the CD38 from MBP, the fusion protein was cleaved by Factor Xa (New England Biolabs), and the CD38 was purified by gel filtration (Sephacryl S-100HR, Pharmacia Biotech, Uppsala, Sweden). To reconstitute the enzymic activity, the purified CD38 was refolded as described (24).…”

Section: Methodsmentioning

confidence: 99%

Lysine 129 of CD38 (ADP-ribosyl Cyclase/Cyclic ADP-ribose Hydrolase) Participates in the Binding of ATP to Inhibit the Cyclic ADP-ribose Hydrolase

Tohgo¹,

Munakata

Takasawa

et al. 1997

CD38 catalyzes not only the formation of cyclic ADPribose (cADPR) from NAD؉ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD ؉ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.Cyclic ADP-ribose (cADPR) 1 (1) induces the release of Ca 2ϩ from microsomes in a variety of tissues and cells including pancreatic ␤ cells (2-10). cADPR is synthesized from NAD ϩ by ADP-ribosyl cyclase, which was purified as a soluble 29-kDa protein from Aplysia ovotestes (11-13). The amino acid sequences of Aplysia ADP-ribosyl cyclases (14, 15) showed a high degree of homology with that of CD38 (16, 17), which was reported to be a surface antigen of human lymphocytes (18). From the experiments in which CD38 cDNA was expressed in mammalian cells, CD38 was shown to catalyze not only the formation of cADPR from NAD ϩ but also the hydrolysis of cADPR to ADP-ribose (ADPR) (19 -21). We have demonstrated that ATP inhibited the hydrolysis, resulting in the accumulation of cADPR (19). Furthermore, we produced transgenic mice overexpressing human CD38 in pancreatic ␤ cells and demonstrated that ATP, produced in the process of glucose metabolism, increased the accumulation of cADPR to enhance the Ca 2ϩ mobilization from the intracellular stores for insulin secretion in the transgenic islets (22).In the present study, we expressed human CD38 in Escherichia coli and purified it to homogeneity. Using the purified CD38, we found that Lys-129 of CD38 participated in cADPR binding and that ATP competed with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38. EXPERIMENTAL PROCEDURESPurification of Soluble CD38 -We isolated a CD38 cDNA from an insulinoma of a Japanese patient by reverse transcriptase-polymerase chain reaction (PCR) (19) and used it for functional analyses of CD38 (17,19,22). The cDNA sequence was exactly the same as the CD38 sequence reported by Jackson and Bell (18) except for two base substitutions (nucleotide 213, A 3 C, and nucleotide 215, C 3 A) (19). The substitutions were also found in the corresponding...