Extracellular Conversion of Epidermal Growth Factor (EGF) to des-Arg53-EGF by Carboxypeptidase M

Abstract

Epidermal growth factor (EGF) is a 53-amino-acid mitogenic polypeptide present in a variety of tissues and fluids including kidney, urine, and amniotic fluid. An EGF isoform, des-Arg53-EGF, has been identified in urine and is the earliest metabolite generated in target cells upon EGF binding. In this study, purified carboxypeptidase M efficiently released the COOH-terminal arginine residue from EGF with a Km = 56 microM, kcat = 388 min-1, and kcat/Km = 6.9 microM-1 min-1. When EGF was incubated with urine or amniotic fluid, des-Arg53-EGF was the only metabolite detected. This conversion was blocked by immunoprecipitation with specific antiserum to carboxypeptidase M or by 10 microM DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (a carboxypeptidase M inhibitor), indicating that the major EGF metabolizing enzyme in these fluids is carboxypeptidase M. When incubated on a confluent monolayer of Madin-Darby canine kidney (MDCK) cells, EGF was readily converted to a single metabolite, des-Arg53-EGF, by carboxypeptidase M. To investigate one possible functional consequence of this conversion, mitogenic activities of EGF and des-Arg53-EGF were tested. Both peptides were equipotent in stimulating [3H]thymidine incorporation in MDCK cells at all doses tested. In addition, inhibition of the conversion of EGF to des-Arg53-EGF by the carboxypeptidase M inhibitor did not affect the mitogenic potency of EGF. These data indicate that carboxypeptidase M, present in a variety of cells and biological fluids, can convert EGF to des-Arg53-EGF. However, in contrast to many other peptide hormones whose activity depends on a final carboxypeptidase processing step, removal of Arg53 of EGF is not required for its mitogenic activity.

Carboxypeptidase M and Kinin B1 Receptors Interact to Facilitate Efficient B1 Signaling from B2 Agonists

“…CPM cleaves only C-terminal Arg or Lys residues, and some of its endogenous substrates include bradykinin, anaphylatoxins C3a, C4a, and C5a, Arg-or Lys-enkephalins, epidermal growth factor, and hemoglobin (2,7,12,13). CPM preferentially cleaves C-terminal Arg as exemplified by the kinetics with Arg 6 -Met 5 -enkephalin (K m ϭ 46 M, k cat ϭ 934 min Ϫ1 ) versus Lys 6 -Met 5 -enkephalin (K m ϭ 375 M, k cat ϭ 663 min Ϫ1 ) (2).…”

Enzymic characterization of a novel member of the regulatory B-like carboxypeptidase with transcriptional repression function: stimulation of enzymic activity by its target DNA

“…The CP activators NiCl # , CoCl # and ZnCl # (CP-N [22], CP-E and CP-D [23], and CP-M [11]) were all found to enhance AEBP1 CP activity. At an activator concentration of 1 mM, a 1.4-fold activation with NiCl # , a 1.6-fold activation with CoCl # and a 1.9-fold activation with ZnCl # was observed after a 20-min assay period (results not shown).…”

“…AEBP1 had a greater affinity for ZnCl # (K a l 0.29 mM) than the other metals, CoCl # (K a l 0.55 mM) and NiCl # (K a l 0.71 mM). The general CP inhibitor and zinc chelator, o-phenanthroline (CP-M [24], CP-N [25], CP-E and CP-D [23], and CP-M [11]), and the CP-specific competitive inhibitor, captopril (CP-N [25]), inhibited AEBP1 CP activity. Captopril (1 µM) reduced AEBP1 CP activity to 54 % after a 20-min assay period, and a concentration of 10 µM completely abolished enzyme activity (results not shown).…”

“…CP-M is a membrane-bound enzyme that removes C-terminal arginine or lysine residues from various polypeptides [8]. McGuire and Skidgel [11] showed that CP-M cleaves epidermal growth factor at the C-terminal arginine to form des-Arg&$-epidermal growth factor. This modified form of epidermal growth factor was found to bind to its receptor better, although the physiological function of this cleavage is unknown.…”

<a name="home"></a>High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

“…The highest levels of CPM have been found in human lung and placenta, but significant amounts are present in kidney, blood vessels, intestine, brain, and peripheral nerves (1,(5)(6)(7). CPM has been found also in soluble form in various body fluids, including amniotic fluid, seminal plasma and urine (5,8,9). Due to its wide distribution in a variety of tissues, it is believed to play important roles in the control of peptide hormone and growth factor activity on the cell surface, and in the membrane-localized degradation of extracellular proteins (10).…”