Internal deletions in human interleukin-6: structure-function analysis.
By cDNA mutagenesis, we have constructed internal and C-terminal deletions (delta 21-51, delta 52-97, delta 97-104, delta 127-174, delta 97-184 and delta 134-184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (delta hIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAb's): mAb's that show preference towards denatured hIL-6, or conformation-specific mAb's. The binding pattern of these two series of mAb's indicated that the IL-6 conformation has been largely destroyed for four of our delta-proteins. Proteins delta 21-51 and delta 127-174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAb's. All of these delta hIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized delta hIL-6 (delta 21-51, delta 127-174, delta 97-184, delta 134-184) did not bind to the IL-6 receptor. Finally, we have produced two proteins with aa 29-33 or 97-104 substituted by corresponding murine IL-6 (mIL-6) sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
Institut Pasteur du Brabant, Bruxelles, Belgium.
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V
Fontaine
J
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Brakenhoff
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De Wit
R
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Arcone
G
G
Ciliberto
J
J
Content
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002474:Cell-Free System; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D015335:Molecular Probes; D008969:Molecular Sequence Data; D011817:Rabbits; D012156:Reticulocytes; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D015850:Interleukin-6; D015335:Molecular Probes