Kinetic Study of Neuropeptide Y (NPY) Proteolysis in Blood and Identification of NPY3–35

Abstract

There is little information on how neuropeptide Y (NPY) proteolysis by peptidases occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive forms and also because the factors affecting the expression of these enzymes have been poorly studied. In the present study, LC-MS/MS was used to identify and quantify NPY fragments resulting from peptidolytic cleavage of NPY Neuropeptide Y (NPY)2 is a 36-amino acid peptide involved in the central and peripheral control of blood pressure (1-4) and in feeding behavior and obesity (5-9). NPY stimulates at least 6 types of receptors, called Y1, Y2, Y3, Y4, Y5, and y6 (10 -12). The Y1 receptor has high affinity for full-length NPY, while Y2 and Y5 receptors bind and are stimulated by fulllength and N-terminally truncated NPY. The physiological effects associated to the Y1 and Y2 receptors are the best known; exposure to a Y1 agonist causes an increase in blood pressure and potentiates postsynaptically the action of other vasoactive substances (1, 4, 13), whereas Y2 receptors are mainly located presynaptically, and upon stimulation mediate the inhibition of neurotransmitter release (14,15). NPY is a prototype of peptide whose function can be altered by proteases. Among peptidases displaying a high affinity for NPY, the primary role appears to be played by dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), a serine-type protease, also known as CD26, that releases an N-terminal dipeptide, Xaa-Xab--Xac, preferentially when Xab is a proline or an alanine residue (16). By cleaving the Tyr-Pro dipeptide off the NPY N-terminal extremity, DPPIV generates NPY 3-36 , a truncated form that loses its affinity for the Y1 receptor and becomes a Y2/Y5 receptor agonist (17, 18).NPY can also be degraded by aminopeptidase P (AmP, EC 3.4.11.9), a metalloprotease that hydrolyzes the peptide bond between the first and the second amino acid residue at the N terminus of proteins, if the second amino acid is a proline (19). AmP removes the N-terminal tyrosine from NPY to generate NPY 2-36 , a selective Y2 agonist (18,20). There is little information on how NPY cleavage by these enzymes occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive (NPYir) forms and also because the factors affecting the expression of these enzymes have been poorly studied. Recently, Frerker et al. (21) reported by MALDI-TOF mass spectrometry that NPY 1-36 is exclusively degraded by DPPIV into NPY 3-36 in EDTA-plasma but they did not provide kinetics of NPY cleavage efficiency of DPPIV. BeckSickinger and co-workers (22) studied with the same technique the metabolic stability of fluorescent N-terminally labeled NPY analogues incubated in human plasma and found that the 36th, 35th, and 33rd residues of NPY analogues may also be removed by unknown carboxypeptidases.We have set up a method using liquid chromatography coupled with tandem mass spectrometry (LC-MS n ) to selectively quantify NPY and its C-terminal fragments NPY 2-36 an...

In vivo and in vitro degradation of peptide YY3–36 to inactive peptide YY3–34 in humans

“…Previous studies have shown that in vitro incubations of NPY in human plasma resulted in cleavage of the COOHterminal amino acid and dipeptide (1,22). We have demonstrated that PYY is significantly degraded to PYY in the pig and also shown that the liver is involved in the COOHterminal truncation (35).…”

“…A method for exact measurements of active PYY 3-36 levels (e.g., sandwich ELISA or LC-MS) would therefore be very valuable in the interpretation of the physiological effects of PYY. In vitro degradation of NPY in human plasma has indicated that NPY is COOH terminally cleaved to primarily NPY , and only trace amounts of NPY have been identified (1). As in pig blood and plasma, PYY was COOH terminally degraded in human blood and plasma in vitro.…”

“…PYY and NPY are COOH terminally amidated and therefore considered relatively protected from COOH-terminal degradation. However, COOH-terminal degradation of NPY, rendering the peptide inactive on the Y1-and Y2-receptors, has been demonstrated in vitro (1,9,22,24). As the sequences of NPY and PYY are 70% homologous, we speculated that PYY might also be prone to COOH-terminal degradation.…”

Mass spectrometry of peptides and proteins from human blood

“…Moreover mass spectrometry has been used to examine peptides without previously understood biological activities (Habermann et al, 2006;Albrethsen et al, 2006b;Aivado et al, 2007). If enzymes are released into, or activated in, the blood and specifically fragment or modify target proteins in a time and dose dependant fashion then these enzyme activities might be assayed using mass spectrometry and pharmacological inhibition (Marshall et al, 2003;Abid et al, 2009;He et al, 2009;Schebb et al, 2009). Biological activities that process blood proteins to release peptides associated with protein regulation might be directly detected by mass spectrometry and independently confirmed by SDS-PAGE and Western blot (Mansfield, 1995;Marshall et al, 2003).…”

Proteomic Identification of Novel Plasma Kallikrein Substrates in the Astrocyte Secretome

“…In addition, PK can function as a plasminogen activator, which could contribute to plasminmediated fibrinolysis, activation of metalloproteinases, and adipocyte differentiation [16,17]. Recently, PK has been shown to cleave neuropeptide Y to generate an inactive fragment [18]. Thus, in addition to PK's well-characterized effects in the intrinsic coagulation pathway and the kallikrein kinin system, these recent findings suggest that PK could have a broader array of substrates that could mediate its effects on vascular physiology and metabolism.…”