Structural, quantitative, and functional characterization of N-glycans in porcine pancreatin extract, lipase, and α-amylase

초록

Porcine pancreatin extract (PPE), an enzyme mixture comprising porcine pancreatic lipase (PPL), porcine pancreatic α-amylase (PPA), and proteases, facilitates the digestion of fats, carbohydrates, and proteins and is widely used to treat pancreatic insufficiency. N-glycosylation is a critical post-translational modification influencing enzyme stability and activity. This study presents the N-glycan profiling of PPE, PPL, and PPA to evaluate their structural roles in enzymatic function. N-glycans were enzymatically released, procainamide-labeled, and analyzed using liquid chromatography–tandem mass spectrometry. Relative quantity of each N-glycan was expressed as a percentage of total N-glycan peak areas, and absolute quantity (pmol) of total N-glycans was calculated using calibration curves. We identified 32 (PPE), 31 (PPL), and 10 (PPA) N-glycans exhibiting diverse structural features, including sialylation and fucosylation. Sialylation was more abundant in PPE (25.6 %) and PPL (29.3 %) than in PPA (10.9 %), whereas fucosylation was prevalent in 67.4 %, 66.9 %, and 54.3 % of structures, respectively. Absolute quantity of total N-glycans was 422 pmol/mg PPE (1.01 pmol/total USP unit), 602 pmol/mg PPL (3.13 × 10−2 pmol/pmol), and 131 pmol/mg PPA (0.72 × 10−2 pmol/pmol). Functional assays after deglycosylation, desialylation, and defucosylation revealed that sialylation and fucosylation influenced the activities of PPE and PPL but had minimal effects on PPA. These results indicate that sialylation and fucosylation are critical for PPE and PPL function but not for PPA. This is the first N-glycan profiling of PPE, PPL, and PPA, and demonstrates the relevance of N-glycans in maintaining structural stability and regulating the enzymatic activity of PPE and PPL.

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