Clusters of basic amino acids in midkine: roles in neurite-promoting activity and plasminogen activator-enhancing activity.
The removal of N-terminally located clusters of basic amino acids (N-tail) or C-terminally located clusters of basic amino acids (C-tail) from the midkine (MK) molecule severely reduced its neurite-promoting activity. However, experiments involving chemically synthesized MK derivatives revealed that the roles of the N-tail and C-tail were mostly indirect ones, i.e. they probably maintain the steric arrangements of the N-terminal and C-terminal halves. In particular, the C-domain, which is the C-terminal half devoid of the C-tail, retained considerable neurite-promoting activity when it was uniformly coated on a dish. The removal of the N-tail or C-tail also reduced the enhancing activity of plasminogen activator (PA) in aortic endothelial cells, although the effect was lower. There are two heparin-binding sites in the C-domain, Clusters I and II. A mutation in Cluster I [R78-->Q] affected the PA-enhancing activity only slightly, and a mutation in Cluster II [K83K84-->QQ] abolished the activity, while both mutations are known to reduce the neurite-promoting activity moderately. Therefore, the two heparin-binding sites in the C-domain play different roles in these two activities. Indeed, heparin exhibited different effects on these two activities. We also observed that intact MK was required for ordered neurite-promotion along the path of MK; one possible interpretation of this is that the N-terminal half is necessary for the stability of the molecule. Furthermore, K76 and K99 were found to be required for the secretion of MK; i.e. mutants in which one of these K residues was changed to Q were produced in the host cells, but not found in the medium.
Akhter
S
S
Department of Biochemistry, Nagoya University School of Medicine, Showa-ku, Nagoya, 466-8550, Japan.
Ichihara-Tanaka
K
K
Kojima
S
S
Muramatsu
H
H
Inui
T
T
Kimura
T
T
Kaneda
N
N
Talukder
A H
AH
Kadomatsu
K
K
Inagaki
F
F
Muramatsu
T
T
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D016207:Cytokines; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051379:Mice; D000078792:Midkine; D009154:Mutation; D009414:Nerve Growth Factors / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects / Q000648:ultrastructure; D010958:Plasminogen / Q000187:drug effects / Q000378:metabolism*; D010960:Plasminogen Activators / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*
D000596:Amino Acids; D002352:Carrier Proteins; D016207:Cytokines; D009414:Nerve Growth Factors; D000078792:Midkine; D010958:Plasminogen; D010960:Plasminogen Activators