Human egasyn binds beta-glucuronidase but neither the esterase active site of egasyn nor the C terminus of beta-glucuronidase is involved in their interaction

Arch Biochem Biophys. 1999 Dec 1;372(1):53-61. doi: 10.1006/abbi.1999.1449.

Abstract

Lysosomal beta-glucuronidase shows a dual localization in mouse liver, where a significant fraction is retained in the endoplasmic reticulum (ER) by interaction with an ER-resident carboxyl esterase called egasyn. This interaction of mouse egasyn (mEg) with murine beta-glucuronidase (mGUSB) involves binding of the C-terminal 8 residues of the mGUSB to the carboxylesterase active site of the mEg. We isolated the recombinant human homologue of the mouse egasyn cDNA and found that it too binds human beta-glucuronidase (hGUSB). However, the binding appears not to involve the active site of the human egasyn (hEg) and does not involve the C-terminal 18 amino acids of hGUSB. The full-length cDNA encoding hEg was isolated from a human liver cDNA library using full-length mEg cDNA as a probe. The 1941-bp cDNA differs by only a few bases from two previously reported cDNAs for human liver carboxylesterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn. The cDNA expressed bis-p-nitrophenyl phosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER. The intracellular hEg coimmunoprecipitated with full-length hGUSB and with a truncated hGUSB missing the C-terminal 18-amino-acid residue when extracts of COS cells expressing both proteins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells. Unlike mEg, hEg was not released from the hEg-GUSB complex with BPNP. Thus, hEg resembles mEg in that it binds hGUSB. However, it differs from mEg in that (i) it does not appear to use the esterase active site for binding since treatment with BPNP did not release hEg from hGUSB and (ii) it does not use the C terminus of GUSB for binding, since a C-terminal truncated hGUSB (the C-terminal 18 amino acids are removed) bound as well as nontruncated hGUSB. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • COS Cells
  • Carboxylic Ester Hydrolases / chemistry
  • Carboxylic Ester Hydrolases / genetics
  • Carboxylic Ester Hydrolases / metabolism*
  • Catalytic Domain
  • DNA Primers / genetics
  • DNA, Complementary / genetics
  • DNA, Complementary / isolation & purification
  • Gene Expression
  • Glucuronidase / chemistry
  • Glucuronidase / genetics
  • Glucuronidase / metabolism*
  • Humans
  • In Vitro Techniques
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Tissue Distribution
  • Transfection

Substances

  • DNA Primers
  • DNA, Complementary
  • Membrane Glycoproteins
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Carboxylic Ester Hydrolases
  • egasyn
  • Glucuronidase