Multiple forms of recombinant murine interleukin-4 expressed in COS-7 monkey kidney cells.
Recombinant murine interleukin-4 (muIL-4) expressed in COS-7 monkey kidney cells was purified to homogeneity by sequential CM-Sepharose, Sephadex G-100 chromatography and mono-S FPLC to a specific activity of 6.10(7) units per mg of protein based on an in vitro HT-2 cell proliferation assay. Two electrophoretic variants, designated a and b, which migrated on SDS-PAGE as a closely spaced doublet with Mr 19,000, were present in the final product. Gas phase sequencing of the purified protein revealed the presence of an N-terminus corresponding to the mature protein predicted from the cDNA sequence and sequencing of a cyanogen bromide digest confirmed 75 of the 120 predicted amino acids. Elution behavior on gel filtration corresponded to that of a monomer of Mr 19,000. Since there are three potential sites of N-glycosylation predicted by the cDNA sequence, the contribution of glycosylation to the observed heterogeneity was examined by treatment with endoglycosidases. Variant b was digested by either endo-beta-N-acetylglucosaminidase H (endo H) or endo-beta-N-acetylglucosaminidase F (endo F) to protein of Mr 15,000 on SDS-PAGE but was unaffected by treatment with endo-beta-N-acetylglucosaminidase D (endo D), thus indicating the presence of high mannose type of N-glycan. In contrast, variant a was resistant to endo H, F and D. Complete conversion of a mixture of variants a and b to a single protein of Mr 15,000 on SDS-PAGE was obtained only after treatment with N-glycanase. Both variants were resistant to neuraminidase and O-glycanase treatment. These data show that the microheterogeneity observed in purified muIL-4 preparations is due to differences in the nature of the N-linked oligosaccharides. The availability of purified recombinant muIL-4 and a methodology for both total and selective deglycosylation provides a basis for the initiation of structure-function studies of this novel T-cell lymphokine.
Ramanathan
L
L
Department of Biotechnology-Biochemistry, Schering Corporation, Bloomfield, NJ 07003.
Le
H V
HV
Labdon
J E
JE
Mays-Ichinco
C A
CA
Syto
R
R
Arai
N
N
Nagabhushan
T L
TL
Trotta
P P
PP
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D015847:Interleukin-4; D007378:Interleukins / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification
D007378:Interleukins; D011994:Recombinant Proteins; D015847:Interleukin-4; D006026:Glycoside Hydrolases