Glycosylation Pattern of Mature Dimeric Leukocyte and Recombinant Monomeric Myeloperoxidase

Abstract

The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro-and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and the threedimensional structure of MPO.Myeloperoxidase (MPO, 5 EC 1.11.1.7) is a heme-containing glycosylated oxidoreductase that catalyzes the production of hypochlorous acid (HOCl) in the presence of hydrogen peroxide (H 2 O 2 ) and chloride anions (1). The macromolecule is a homodimer with each monomer consisting of a light and a heavy chain. The dimer is stabilized by a disulfide bridge and by several interactions between the carbohydrate moieties located at the dimer interface (2). MPO is a lysosomal enzyme present in the azurophilic granules of human neutrophils and monocytes and is synthesized in the promyelocytic state during maturation of the polymorphonuclear (PMN) leukocytes (3).During phagocytosis of exogenous structures, neutrophils release their granule content (e.g. MPO) into the phagosome. Respiratory burst is initiated by activation of NADPH-oxidase that reduces molecular oxygen and releases superoxide radical anions. Upon superoxide dismutation, hydrogen peroxide is produced that mediates MPO-driven oxidation of chloride to antimicrobial HOCl.More recent data have indicated that these reactions are not restricted to the phago-lysosome. Indeed, under oxidative stress (i.e. excessive and uncontrolled production of various reactive oxygen-derived species, ROS), MPO can escape from neutrophils and circulate in extracellular fluids. Thereby it might cause oxidative modification to host tissues and biomolecules. The potential deleterious effect of this "circulating" MPO has been demonstrated in various pathological conditions involving chronic inflammation, such as atherosclerosis, demyelinating disorders of the central nervous system or endstage renal disease (4 -9).In many of these experiments, recombinant human MPO (r-MPO) expressed in a Chinese hamster ovary (CHO) cell line has been used (10) because recombinant production delivers higher amounts of protein as compared with the classical purification from neutrophils (11). However, although having the same catalytic activity as the dimeric leukocyte enzyme (h-MPO) (12-14), biosynthesis of r-MPO in CHO cell lines lacks some proteolytic steps. As a consequence, r-...

Deglycosylation influences the oxidation activity and antigenicity of myeloperoxidase

“…As a peroxidase, the most vital physiological function of MPO is determined by its enzymatic activity, to catalyze the production of hypochlorous acid and reactive oxygen species, and to participate in the antimicrobial mechanism in circulation and at sites of inflammation . A recent study indicated glycosylation as being critical to the enzymatic activity of MPO; our results are consistent in this respect. We further investigated the effect of each glycan on the oxidation function of MPO.…”

“…The mature MPO protein consists of two light and two heavy polypeptide chains with each heavy chain binding to prosthetic heme group . Mass spectrometry has demonstrated the presence of five N‐linked glycosylation sites in each MPO heavy chain, which are occupied by either complex or high mannose glycan structures . The overall structures of MPO glycan and the biological effects of glycosylation patterns on MPO function are not well characterized.…”

Low-Density Lipoprotein Modified by Myeloperoxidase in Inflammatory Pathways and Clinical Studies

“…MPO also contains a calcium binding site contributing to the stabilization of the structure. N-glycans play a key role in protein synthesis and also in enzymatic activity as recently shown by our experiments [52]. Furthermore, MPO is a highly cationic protein with a pI ≈ 11 enabling its binding to electronegative surfaces such as endothelial wall, lipoprotein, or proteoglycans [53, 54].…”

Multidomain Human Peroxidasin 1 Is a Highly Glycosylated and Stable Homotrimeric High Spin Ferric Peroxidase

“…This matches with five N-glycans in mature homodimeric MPO and pro-MPO produced in CHO cell lines (21). Monomeric bovine lactoperoxidase has four N-glycosylation sites (38).…”

“…For N-glycan site occupancy, a rapid resolution liquid chromatographic system (1200 series) coupled to a quadrupole/ time-of-flight equipped with an electrospray ionization source (ESI-QTOF 6520 series) mass spectrometer (Agilent Technologies, Palo Alto, CA) was used for analysis of the aforementioned protein digests (21). As PNGase F removes glycans and performs deamination of asparagine (Asn 3 Asp), the glycosylation occupancy was calculated by measuring the ratio between the area under the curve of the native peptide and its deaminated form.…”