Thyroglobulin processing by thyroidal proteases. Major sites of cleavage by cathepsins B, D, and L

J Biol Chem. 1991 Oct 25;266(30):20198-204.

Abstract

The normal provision of thyroid hormones to the body requires their release from the prohormone, thyroglobulin (Tg). Previous work established the importance of cathepsins B, D, and L (formerly designated cysteine proteinase I) to this process but had not defined the points of proteolytic attack for each enzyme. In the present study we labeled rabbit Tg in vivo with sodium 125I and performed limited digestions with cathepsins B, D, and L, purified from human thyroids. The resultant peptide fragments were analyzed by amino-terminal sequencing and located within the Tg molecule by comparison with the cDNA-derived sequences from human Tg. We identified three cleavage points for cathepsin B, corresponding to P'1 residues 532, 795, and 2487; four cleavage points for cathepsin L, corresponding to P'1 residues 2389, 2452, 2490, and 2657; and four cleavage points for cathepsin D, corresponding to P'1 residues 551, 1835, 2468, and 2643. None of the cleavage points was near Tgs known hormonogenic sites, but these peptide fragments contained three of the four major hormonogenic sites in rabbit Tg, suggesting some preference for their early proteolytic processing. Cathespin B alone among the three endopeptidases had some exopeptidase activity toward Tg. The cleavage specificities for each of the endopeptidases resembled those described with other protein substrates. Thus, cathepsin D preferentially cleaved bonds between hydrophobic residues, and cathespin L cleaved bonds with hydrophobic residues at P2 and P3. Although cathepsin Bs specificity was less obvious, it produced a major cleavage between 2 leucine residues. The existence of three endopeptidases cleaving at different sites shows that Tg proteolysis is a complex process, suggests synergism among their enzyme activities, and provides a physiological mechanism for selective hormone release, including its regulation by TSH.

Publication types

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

MeSH terms

  • Alkylation
  • Amino Acid Sequence
  • Animals
  • Cathepsin B / metabolism*
  • Cathepsin D / metabolism*
  • Cathepsin L
  • Cathepsins / metabolism*
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Cysteine Endopeptidases
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases*
  • Hydrolysis
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Rabbits
  • Thyroglobulin / metabolism*

Substances

  • Thyroglobulin
  • Cathepsins
  • Endopeptidases
  • Cysteine Endopeptidases
  • Cathepsin B
  • CTSL protein, human
  • Cathepsin L
  • Cathepsin D