Identification of critical amino acid residues in human and mouse granulocyte-macrophage colony-stimulating factor and their involvement in species specificity

A.B Shanafelt,K.E Johnson,R.A Kastelein

doi:10.1016/s0021-9258(18)92772-9

Abstract

Segments critical to the activity of human granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified by scanning deletion analysis and compared with the critical regions previously identified in the homologous mouse GM-CSF protein. Three of the four critical regions thus identified are in equivalent positions in their respective polypeptides, while a fourth critical region of each is uniquely located. To investigate whether unique critical regions are responsible for the observed species specificity of human and mouse GM-CSF, all critical regions were substituted into their opposite homologue. This identified one specific, but different, critical region in each homologue that could not be replaced. Further characterization of the nature of the species specificity of these two proteins was accomplished by the generation of a series of human/mouse GM-CSF hybrids. Each hybrid protein was assayed for specific activity on human- and mouse GM-CSF-dependent cell lines. Significant differences in the specific activity of these hybrids was observed, suggesting that different segments of each molecule interact with their respective receptors. Based on these two approaches, individual amino acids were identified that could provide, at least in part, the interactions between these protein ligands and their respective receptors. These residues are Thr-78 and Met-80 in human GM-CSF and Asp-92, Thr-98, and Asp-102 in mouse GM-CSF.

Highlights

MATERIALS ANDMETHODS in the specific activity of these hybrids was observed, Bacterial Host Strains and Vectors-Escherichia coli K12 strain suggesting that different segments of each molecule JMlOl [14] was used as the hostfor propagation and maintenanceof interact with their respective receptors
We have described the expression of biologically active, matureGM-CSFwiththisE. coli secretory expression system[18].ThehumanGM-CSF
Despite a follows: GM-CSF cDNAsequences were oriented in tandem, sepahigh level of amino acid homology and similarphysical char- rated by EcoRI and SacI restriction sites, in pINIIIompH3 [17], and acteristics [7, 8],the two polypeptides are species specific at both thebiological and receptor bindinglevels [6].’

Summary

Systematic Introduction of Deletions Throughout Human

GM-CSF-The structuraland/orfunctionalimportance of IV specific regions within the human GM-CSFpolypeptide were Human P~TPETSCATQYITFESFKENYKDFLLVIP~DCWWVQE examined by the systematic introduction of deletions along Mouse PPT~gTDCETQVTT~~ADFIDSLKT~~oLTDIPFECK~~~PSOK the entire length of the molecule. 56 deletion proteinswith respect tothe location of their relative positions for critical regions I, 111, and IV are comdeletions. Any mutant protein exhibitingless than 0.01% of wild- Substitution of Critical Regions Has Different Effects on typeactivity was consideredinactive, andtheamino acid Human andMouse GM-CSF-The effect of individual critical residues in the corresponding deletion mutant were referred regions on the activity of human and mouse GM-CSF was to as critical to the activitoyf human GM-CSF. All mutants were assayed on both the TF1 and tified in humanGM-CSFcomparedwiththose previously NFS6O cell lines, and relative activity to human GM-CSF HM2,mouse critical region IIm substituted generated by eitherin uiuo recombinationorsite-directed into thecorresponding region of human GM-CSF; mH2, the parallel mutant in mouse GM-CSF. All mutant polypeptides were assayed onboththe TF1 and NFSGO cell lines and relative activity to human GM-CSF (in tTheF1 assay) ormouse GMCSF (in theNFSGO assay) was determined (Table 11)

Human critical regions in mouse

Acid ResidHuuesmiann

Two other lineosf evidence support the notion threastidues

DISCUSSION