Towards characterization of the glycoproteome of tomato (Solanum lycopersicum) fruit using Concanavalin A lectin affinity chromatography and LC-MALDI-MS/MS analysis

PROTEOMICS ◽  
2011 ◽  
Vol 11 (8) ◽  
pp. 1530-1544 ◽  
Author(s):  
Carmen Catalá ◽  
Kevin J. Howe ◽  
Simon Hucko ◽  
Jocelyn K. C. Rose ◽  
Theodore W. Thannhauser
Keyword(s):  
Affinity Chromatography ◽  
Solanum Lycopersicum ◽  
Concanavalin A ◽  
Maldi Ms ◽  
Lectin Affinity Chromatography ◽  
Lectin Affinity ◽  
Ms Analysis

scholarly journals A Retrospective Analysis of the Cartilage Kunitz Protease Inhibitory Proteins Identifies These as Members of the Inter-α-Trypsin Inhibitor Superfamily with Potential Roles in the Protection of the Articulatory Surface

2019 ◽  
Vol 20 (3) ◽  
pp. 497 ◽  
Author(s):  
Susan Smith ◽  
James Melrose
Keyword(s):  
Articular Cartilage ◽  
Affinity Chromatography ◽  
Trypsin Inhibitor ◽  
Concanavalin A ◽  
Inhibitory Activity ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Lectin Affinity Chromatography ◽  
Joint Function ◽  
Lectin Affinity

Aim: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. Methods: Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion. Results: 2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function. Discussion/Conclusions: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.

scholarly journals A Retrospective Analysis of the Cartilage and Intervertebral Disc Kunitz Protease Inhibitory Proteins Identifies These as Members of The Inter-α-Trypsin Inhibitor Superfamily with potential roles in the protection of the articulatory surface

2018 ◽  
Author(s):  
Susan Smith ◽  
James Melrose
Keyword(s):  
Articular Cartilage ◽  
Affinity Chromatography ◽  
Trypsin Inhibitor ◽  
Concanavalin A ◽  
Inhibitory Activity ◽  
Proteinase Inhibitors ◽  
Cathepsin G ◽  
Prolonged Storage ◽  
Lectin Affinity Chromatography ◽  
Lectin Affinity

The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. Ovine articular cartilage was finely diced and extracted in 6M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity and pooled fractions assessed by affinity blotting using biotinylated trypsin to detect active SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS stub epitope generated by chondroitinase-ABC digestion. This identified 2-B-6 (+) positive 220-250,120, 58 and 36 kDa SPIs. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulphate stub epitope consistent with its identity as the α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa could be autolytically generated by prolonged storage of the aforementioned 120 and 58 kDa SPIs; chymotrypsin affinity chromatography also generated the 6kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 both displayed potent inhibitory activity towards trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and HA in the surface regions of articular cartilage represented probable binding sites for the ITI SPs with likely importance in the preservation of joint function.

scholarly journals Characterization of Glycoproteins in Pancreatic Cyst Fluid Using a High-Performance Multiple Lectin Affinity Chromatography Platform

2013 ◽  
Vol 13 (1) ◽  
pp. 289-299 ◽  
Author(s):  
Francisca Owusu Gbormittah ◽  
Brian B. Haab ◽  
Katie Partyka ◽  
Carolina Garcia-Ott ◽  
Marina Hancapie ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
High Performance ◽  
Pancreatic Cyst ◽  
Cyst Fluid ◽  
Lectin Affinity Chromatography ◽  
Lectin Affinity ◽  
Pancreatic Cyst Fluid
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