Circular permutation of the granulocyte colony-stimulating factor receptor agonist domain of myelopoietin.
Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric proteins with therapeutic potential.
McWherter
C A
CA
Searle Discovery Research, Monsanto Company, St. Louis, Missouri 63198, USA. charles.a.mcwherter@monsanto.com
Feng
Y
Y
Zurfluh
L L
LL
Klein
B K
BK
Baganoff
M P
MP
Polazzi
J O
JO
Hood
W F
WF
Paik
K
K
Abegg
A L
AL
Grabbe
E S
ES
Shieh
J J
JJ
Donnelly
A M
AM
McKearn
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002942:Circular Dichroism; D016179:Granulocyte Colony-Stimulating Factor; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D006801:Humans; D007377:Interleukin-3; D017997:Leukotriene C4 / Q000097:blood / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000819:agonists* / Q000235:genetics* / Q000378:metabolism; D016185:Receptors, Interleukin-3 / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000138:chemical synthesis* / Q000378:metabolism; D011994:Recombinant Proteins; D020349:Surface Plasmon Resonance
D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D010446:Peptide Fragments; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D016185:Receptors, Interleukin-3; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C404145:myelopoietin; D016179:Granulocyte Colony-Stimulating Factor; D017997:Leukotriene C4