Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin.
Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity.
Rose
Christiane
C
Unité de Neurobiologie et Pharmacologie Moléculaire (U.109) de l'INSERM, Centre Paul Broca, 2ter rue d'Alésia, 75014 Paris, France.
Voisin
Stéphanie
S
Gros
Claude
C
Schwartz
Jean-Charles
JC
Ouimet
Tanja
T
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D051381:Rats; D013379:Substrate Specificity
D007527:Isoenzymes; D015260:Neprilysin