Enzymatical hydrolysis of N-glycans from glycoproteins and fluorescent labeling by 2-aminobenzamide (2-AB).

When performing a structural analysis of N-glycans, a number of aspects should be considered. N-Glycans may be hydrolyzed from purifi ed glyc-oproteins, serum glycoprotein mixtures, or delipidated membrane fractions by chemical hydrolysis using hydrazine or enzymatic hydrolysis using PNGase F. Chemical deglycosylation may be an economical alternative to produce N-and O-glycans in a preparative scale, but it is less suitable for analytical purposes. By chemical hydrazinolysis the protein core is destroyed completely and all acyl groups are cleaved from neuraminic acid residues as well as from N-acetylhexosamine residues. If not only a structure analysis of N-glycans is intended but a sequencing of the protein core, an analysis of the different types of neuraminic acids or an elucidation of the carbohydrate structures in distinct glycosylation sites has to be performed in addition, enzymatical deglycosylation using PNGase F is the most suitable way to hydrolyze N-glycans from the protein backbone.

The insect metalloproteinase inhibitor gene of the lepidopteran Galleria mellonella encodes two distinct inhibitors.

The insect metalloproteinase inhibitor (IMPI) from the greater wax moth, Galleria mellonella, represents the first and to date only specific inhibitor of microbial metalloproteinases reported from animals. Here, we report on the characterization including carbohydrate analysis of two recombinant constructs encoded by impi cDNA either upstream or downstream of the furin cleavage site identified. rIMPI-1, corresponding to native IMPI purified from hemolymph, is encoded by the N-terminal part of the impi sequence, whereas rIMPI-2 is encoded by its C-terminal part. rIMPI-1 is glycosylated at N48 with GlcNAc2Man3, showing fucosylation to different extents. Similarly, rIMPI-2 is glycosylated at N149 with GlcNAc2Man3, but is fully fucosylated. rIMPI-1 represents a promising template for the design of second-generation antibiotics owing to its specific activity against thermolysin-like metalloproteinases produced by human pathogenic bacteria such as Vibrio vulnificus. In contrast, rIMPI-2 does not inhibit bacterial metalloproteinases, but is moderately active against recombinant human matrix metalloproteinases (MMPs). Both microbial metalloproteinases and MMPs induce expression of the impi gene when injected into G. mellonella larvae. These findings provide evidence that the impi gene encodes two distinct inhibitors, one inhibiting microbial metalloproteinases and contributing to innate immunity, the other putatively mediating regulation of endogenous MMPs during metamorphosis.