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gC1q Fragment Isolation Protocol
The gC1q fragment can be obtained by collagenase digestion of the collagen-like fragment of purified C1q. The collagenase and undigested C1q is removed from the incubation mixture by size-exclusion chromatography.
Fig.1 Flow chart of gC1q fragment isolation protocol by collagenase digestion. (Creative Biolabs)
Published Data
Fig.2 Mapping of potential neutrophil elastase cleavage sites on gC1q.1
To experimentally pinpoint the exact cleavage sites, a mutagenesis analysis was employed. Initially, the main products from neutrophil elastase (NE) digestion of gC1q, with apparent molecular weights between 12 and 17 kDa, were gel-purified and analyzed using MALDI-TOF. Two primary peaks, with molecular weights of 8650.7 and 8646.3 Da, closely matched virtual fragments with calculated masses of 8651 Da for cleavage between S913 (P1) and R914 (P1’) and 8647 Da for cleavage between L912 (P1) and S913 (P1’). NMR models revealed these potential cleavage sites were solvent-exposed. To disrupt NE recognition, several gC1q domain mutants were generated at R914 and S913. Their proper folding was verified through solubility and thermostability assays before undergoing NE digestion.
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Reference
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Maiorani, Orlando, et al. "Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4β1 integrin interaction, cell adhesion and anti-proliferative activity." Scientific Reports 7.1 (2017): 39974. Distributed under Open Access license CC BY 4.0, without modification.
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