Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor.
By using probes based on partial amino acid sequence of glycosylation-inhibiting factor (GIF) from a mouse T-cell hybridoma, a full-length cDNA encoding mouse GIF was isolated. A cDNA clone encoding human GIF was isolated from cDNA libraries of a GIF-producing human T-cell hybridoma by using mouse GIF cDNA as a probe. The cDNAs encode a putative 12.5-kDa peptide of 115 amino acids. Northern blot analysis demonstrated a single, 0.6-kb transcript. Polyclonal rabbit antibodies against the Escherichia coli-derived recombinant 13-kDa peptide bound hybridoma-derived GIF. Although the peptide did not contain a signal peptide sequence, transfection of the cDNA into COS-1 cells resulted in secretion of 13-kDa peptide, but the peptide had substantially less bioactivity than the hybridoma-derived GIF. However, expression of a chimeric cDNA encoding a fusion protein consisting of the N-terminal pro region of calcitonin precursor and human GIF and cotransfection with furin cDNA to allow intracellular cleavage of the fusion protein resulted in secretion of 13-kDa peptide that was comparable to hybridoma-derived GIF in its bioactivity. Both the 13-kDa peptide and GIF bioactivity in the transfected COS-1 supernatant bound to a monoclonal antibody against hybridoma-derived human GIF. These results indicate that the 13-kDa peptide represents recombinant GIF, but posttranslational modification of the peptide is important for generation of the bioactivity. The GIF cDNA had high homology with the cDNA encoding macrophage migration inhibitory factor. However, the recombinant GIF failed to inhibit migration of human monocytes, and recombinant human macrophage migration inhibitory factor did not have GIF bioactivity.
Mikayama
T
T
Division of Immunobiology, La Jolla Institute for Allergy and Immunology, CA 92037.
Nakano
T
T
Gomi
H
H
Nakagawa
Y
Y
Liu
Y C
YC
Sato
M
M
Iwamatsu
A
A
Ishii
Y
Y
Weiser
W Y
WY
Ishizaka
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D045683:Furin; D015870:Gene Expression; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008222:Lymphokines / Q000096:biosynthesis* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D029584:Prostatic Secretory Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013381:Subtilisins / Q000096:biosynthesis; D013491:Suppressor Factors, Immunologic / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D008222:Lymphokines; D029584:Prostatic Secretory Proteins; D011994:Recombinant Proteins; D013491:Suppressor Factors, Immunologic; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors; D013381:Subtilisins; D045683:Furin