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Abstract: The neutrophil granulocyte is an important first line of defense against intruding pathogens and it contains a range of granules armed with antibacterial peptides and proteins. Proteinase 3 (PR3) is one among several serine proteases of the azurophilic granules in neutrophil granulocytes. Here, we characterize the glycosylation of PR3 and its association with antimicrobial human neutrophil peptides (HNPs, α-defensins) and the effect of these on the mechanism of inhibition of the major plasma inhibitor of PR3, α1-antitrypsin. The glycosylation of purified, mature PR3 showed some heterogeneity with carbohydrates at Asn 102 and 147 carrying unusual small moieties indicating heavy processing. Mass spectrometric analysis and immuno blotting revealed strong association of highly purified PR3 with α-defensins and oligomers hereof. Irreversible inhibition of PR3 by α1-antitrypsin did not affect its association with defensins. Other proteins from neutrophil granules were also found to be associated with defensins, whereas purified plasma proteins did not carry defensins. These results point to a role of defensins in controlling and targeting the activity of neutrophil granule proteins. [Copyright &y& Elsevier]

Abstract: The enzyme myeloperoxidase (MPO) is an important part of the neutrophil immune reaction and can be found in alfa granula. The presence of MPO can be used to distinguish acute myelogenous leukemia from acute lymphocytic leukemia. However, the methods employed to do so, such as flow cytometry and immunohistochemistry rely on antibody recognition, and therefore the characterization of the mature MPO, including post-translational modifications, must be considered as important as epitope mapping. MPO has 5 N-linked glycosylation sites, occupied by both high mannose and complex glycan structures. In this study we utilize intact glycopeptide MSMS analysis for site specific characterization of the glycan structures of MPO from a cancer patient. The identified glycan structures are compared to those of MPO from healthy donors, in order to probe for any potential differences that may have diagnostic use. [ABSTRACT FROM AUTHOR]

Abstract: The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood. [Copyright &y& Elsevier]