Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue

Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11054-61. doi: 10.1073/pnas.96.20.11054.


Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a "fold-specific" inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription-PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • Enzyme Activation
  • Humans
  • Kinetics
  • Male
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Prostate / enzymology*
  • Prostatic Neoplasms / enzymology*
  • Prostatic Neoplasms / etiology
  • RNA, Messenger / analysis
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / isolation & purification*
  • Serine Proteinase Inhibitors / pharmacology*
  • Substrate Specificity


  • RNA, Messenger
  • Serine Proteinase Inhibitors
  • Serine Endopeptidases

Associated data

  • GENBANK/AF133086