Limited proteolysis by chymotrypsin of midkine and inhibition by heparin binding

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Y Matsuda1, A H Talukder, M Ishihara

  • 1Department of Biochemistry, Nagoya University School of Medicine, Japan.

Biochemical and Biophysical Research Communications
|November 1, 1996
PubMed

Insights

Heparin specifically inhibits chymotrypsin digestion of midkine (MK), revealing its two-domain structure and a stabilizing N-domain. This interaction requires at least six sugar units and sulfonyl groups on heparin.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Glycobiology

Background:

  • Midkine (MK) is a growth factor involved in various biological processes.
  • Understanding MK's structural properties and interactions is crucial for its functional characterization.
  • Proteolytic digestion can reveal structural domains and functional sites within proteins.

Purpose of the Study:

  • To investigate the limited proteolysis of midkine (MK) by chymotrypsin.
  • To identify the specific inhibitors and structural requirements for this proteolytic event.
  • To elucidate the domain structure and potential stabilizing mechanisms of MK.

Main Methods:

  • Limited proteolysis of MK using chymotrypsin.
  • Analysis of proteolytic fragments by molecular weight (Mr).
  • Inhibition assays using heparin and its derivatives (oligosaccharides, desulfated heparin).
  • Identification of the cleavage site (Phe55).

Main Results:

  • Chymotrypsin digestion yielded two MK fragments (11,000 and 6,000 Da) via cleavage at Phe55.
  • Heparin specifically inhibited this proteolysis, while other glycosaminoglycans did not.
  • A minimum of 6 heparin monosaccharide units and sulfonyl groups were essential for inhibition.
  • MK possesses distinct N- and C-domains, with Phe55 located in an exposed hinge region.

Conclusions:

  • Midkine (MK) has a two-domain structure (N- and C-domains) separated by a proteolytically sensitive hinge region.
  • Heparin binding, requiring specific structural features, protects MK from chymotrypsin degradation.
  • The N-domain may confer stability to the C-domain in the intact MK molecule.

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