Synthetic chimeras of mouse growth factor-associated glandular kallikreins. II. Growth factor binding properties.
Six chimeric constructs of the sequentially similar growth factor-associated kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--have been expressed, and their ability to generate complexes with epidermal growth factor (EGF) and beta-NGF, analogous to the high molecular weight forms (7S NGF and HMW-EGF) found in the mouse submaxillary gland, evaluated. The chimeras are distinguished by the interchange of three regions composing the amino, middle, and carboxyl terminal regions that encompass four surface loops possibly involved in specific growth factor interactions. Native beta-NGF (along with native alpha-NGF) formed complexes indistinguishable from naturally occurring 7S NGF, characterized by an alpha 2 beta gamma 2 structure (where beta-NGF is itself a dimer), with recombinant (r) gamma-NGF and with a chimera in which the amino terminal region from EGF-BP was substituted. Two other chimeras containing either the middle or carboxyl terminal regions of gamma-NGF showed weaker ability to form 7S complexes. Thus, all chimeras containing two segments from gamma-NGF retained at least some ability to form the 7S complex. rEGF-BP reacted weakly with EGF, but the chimera composed of the amino and middle segments of EGF-BP and the carboxyl terminal segment of gamma-NGF formed a nativelike HMW-EGF complex. None of the other chimeras appeared to bind EGF. These results identify amino acid positions within each kallikrein that participate in strong growth factor interactions and demonstrate that, outside of active site contacts, different regions of the kallikreins are involved in the binding of EGF and beta-NGF, respectively.
Blaber
M
M
Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717.
Isackson
P J
PJ
Holden
H M
HM
Bradshaw
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D007610:Kallikreins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D020840:Tissue Kallikreins
D046911:Macromolecular Substances; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D007610:Kallikreins; C489120:Klk13 protein, mouse; D020840:Tissue Kallikreins; C489121:Klk1b22 protein, mouse; C489119:Klk9 protein, mouse; C053223:epidermal growth factor binding protein, mouse