Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor.

Abstract

By using probes based on partial amino acid sequence of glycosylation-inhibiting factor (GIF) from a mouse T-cell hybridoma, a fufl-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. Polydonal rabbit antibodies against the Escherichia cofi-derived recombinant 13-kDa peptide bound hybridoma-derived GIF. Although the peptide did not contain a signal peptide sequence, tansfection of the cDNA into COS-1 celis resulted in secretion of 13-kDa peptide, but the peptide had substantially less bioactivity than the hybridoma-derived GIF. However, expression ofa 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 intraceflular 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 tansfected 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.

Inhibition of macrophage migration inhibitory factor (MIF) tautomerase and biological activities by acetaminophen metabolites

“…It is noteworthy that no inhibition of MIF enzymatic activity was obtained with the thiol-reactive agents iodoacetamide (17) and N-ethylmaleimide (18), suggesting that NAPQI and HQ are reacting at sites other than free cysteines present in MIF. The efficiency of MIF enzyme inhibition by the quinones varied according to the concentration of MIF present in the reaction.…”

“…MIF has the capacity to override, or counterregulate, the immunosuppressive effects of glucocorticoids (5,6,8,10), which accounts in part for the proinflammatory role of MIF in conditions such as septic shock (5,6,(11)(12)(13), arthritis (14)(15)(16), and glomerulonephritis (17). Additional biological activities for MIF include the regulation of macrophage and T cell activation (7,8), IgE synthesis (18), insulin release (19) and carbohydrate metabolism (20,21), cell growth and apoptosis (22,23), and tumor angiogenesis (24).…”

A Diversified Family of 12‐kDa Proteins with a High Amino Acid Sequence Similarity to Macrophage Migration‐Inhibitory Factor (MIF)

“…The genomic sequence of hMIF was recently elucidated by Paralkar and Wistow (1994) and the sequence is identical to that encoded by the cDNA of hGIF which was derived from T-cells by Mikayama et al (1993). It remains to be established if the cDNA sequence of hMIF given by Weiser et al (1989) is an isoform of hGIF or is due to a sequencing error.…”

“…The cDNAs encoding MIF-like proteins were also isolated from a chicken embryonic eye lens library (Wistow et al, 1993) and a mouse cDNA library in the class of delayed early response genes (Lanahan et al, 1992). The latter appears to have identical amino acid sequence to murine glycosylation-inhibiting factor (GIF) which has the ability to switch normal T-cells from production of glycosylated IgE-binding-factor complex to formation of the unglycosylated complex (Mikayama et al, 1993;Gomi et al, 1994). We have recently isolated two isoforms of a 12-kDa protein (pl2" and ~1 2~) from calf brain tissues and showed that their N-termini share a high sequence similarity with that of human (h)MIF .…”

CD4 cell-secreted, posttranslationally modified cytokine GIF suppresses Th2 responses by inhibiting the initiation of IL-4 production

“…GIF is encoded by the same gene that codes for macrophage migration inhibitory factor (MIF) (5). It is highly conserved throughout the animal kingdom although it does not belong to any cytokine gene family.…”