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

Lysosomal beta-glucuronidase shows a dual localization in mouse liver, wher e a significant fraction is retained in the endoplasmic reticulum (ER) by i nteraction with an ER-resident carboxyl esterase called egasyn, This intera ction 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 (hGU SB), However, the binding appears not to involve the active site of the hum an 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 li brary using full-length mEg cDNA as a probe. The 1941-bp cDNA differs by on ly a few bases from two previously reported cDNAs for human liver carboxyle sterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn, The cDNA expressed bis-p-nitrophenyl p hosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER, The intracellular hEg coimmunoprec ipitated with full-length hGUSB and with a truncated hGUSB missing the C-te rminal 18-amino-acid residue when extracts of COS cells expressing both pro teins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells, Unlike mEg, hEg was no t 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 a ppear 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 ami no acids are removed) bound as well as nontruncated hGUSB, Evidence is pres ented that an internal segment of 51 amino acids between 228 and 279 residu es contributes to binding of hGUSB by hEg. (C) 1999 Academic Press.