The Biological Activity of a Novel PhotoaffinityLabelled Imino Sugar Jane Atkin St. Hilda’s College.
Download ReportTranscript The Biological Activity of a Novel PhotoaffinityLabelled Imino Sugar Jane Atkin St. Hilda’s College.
The Biological Activity of a Novel Photoaffinity Labelled Imino Sugar
Jane Atkin St. Hilda’s College
OVERVIEW
Background Methods Results & Discussion Acknowledgements Questions
BACKGROUND (1)
Glc Glc Glc Man Man Man Man Man Variable Core Man Man Man Man GlcNAc GlcNAc NH 3 + ---X-Asn-X-(Ser/Thr)---COO Glycoprotein formation: transfer of an oligosaccharide from a lipid precursor to a polypeptide Processing of this glycan region – variable residues Endoplasmic reticulum and Golgi
BACKGROUND (2)
BACKGROUND (3)
Terminally misfolded proteins are broken down by ER-
associated protein degradation (ERAD)
Retrotranslocation, then deglycosylation Protein moiety is ubiquitinated and broken down by the proteasome Free oligosaccharides (FOS) broken down in the lysosome
Sec61 ER lumen Cytosol Misfolded glycoprotein PNGase Proteasome
BACKGROUND (4)
NB-DNJ is an inhibitor of α -glucosidases I and II Inhibition monitored by glucosylated FOS build-up Potential for anti-viral therapy: HIV, Hepatitis B
BACKGROUND (5)
DNJ-nitro-phenyl azide (DNJ-NAP) made to investigate the behaviour of NB-DNJ Cross-links to neighbouring amide groups upon exposure to short-wave ultraviolet (UV) light
20-100-fold better inhibitory activity than NB DNJ on α -glucosidases in the cell
AIMS
To determine the biological activity of this DNJ-NAP To observe the interactions this DNJ-NAP compound makes during its time in the cell by incubating it with cell samples for different time periods before exposure to UV light
METHODS
Produced NB-DNJ derivative by reacting DNJ with the aldehyde linker Aldehyde Linker Reductive Amination DNJ DNJ-NAP Compared its inhibitory activity to that of an existing version of the compound and NB-DNJ, by incubating with HL60 cells and analysing the resulting glucosylated free oligosaccharides
RESULTS
Build-up of glucosylated FOS My compound (NAP) is slightly more potent than previously synthesised compound (GS)
IN VITRO
EXPERIMENTS (1)
Radiolabelled the photo-activatable inhibitor with 3 H Bovine Serum Albumin (BSA) in vitro experiments: • Denatured the protein 1. Added 3 H-DNJ-NAP to BSA and exposed to UV to see if it could react in the cell 2. Ran on SDS gel 3. Measured radioactivity of gel slices using scintillation counter • No radioactivity associated with protein • Suggests BSA and DNJ-NAP do not interact – i.e. cross linking is not random
IN VITRO
EXPERIMENTS (4)
Therefore, repeated experiment with glucosidase enzymes – α , β Also, crude rat liver extract to investigate how selective the binding is Analysed using a radioactivity detector plate Could not see any radioactivity associated with protein bands in the gels
IN VITRO
EXPERIMENTS (5)
Results e.g. β -glucosidase (impure) Same for liver extract and α -glucosidase β-glucosidase + 3 H-DNJ NAP + UV
Free radioactivity
CELLULAR EXPERIMENTS (1)
Incubated light 3 H-DNJ-NAP with HL60 cells for different time periods before exposure to UV 0, 1, 2, 4, 10, 30, 60 minutes Control: 60 minute incubation but no UV exposure 2-day long-term experiment
CELLULAR EXPERIMENTS (2)
Again, no radioactivity could be observed using the screen Tried new technique Cut up dried gels into ~2mm slices and measured the radioactivity using scintillation counter Results promising…
RESULTS
All radioactivity was free and not associated with protein from 0-4 minutes
Radioactivity of Gel Slices From 4 Minute Incubation
300 250 200 150 100 50 0 0 2 4 6 8 10 12 14 16 18 20 22
Slice Number
24 26 28 30 32 34 36 38
RESULTS (2)
After 10 minutes, radioactivity was associated with a protein More binding after 30 minutes (~4x higher cpm) 250
Radioactivity of Gel Slices From 10 Minute Incubation
200 150 100 50 0 0 2 4 6 8 10 12 14 16 18 20 22
Slice Number
24 26 28 30 32 34 36 38 40
Radioactivity of Gel Slices From 30 Minute Incubation
900 800 700 600 500 400 300 200 100 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26
Slice Number
28 30 32 34 36 38 40 42 44 46
RESULTS (3)
After 60 minutes, there is no longer any radioactivity associated with protein. Also seen after two days
Radioactivity of Gel Slices from 60 Minute Incubation
60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 18 20 22
Slice Number
24 26 28 30 32 34 36 38 40
RESULTS (4)
Used this technique to analyse β -glucosidase gel No radioactivity associated with protein May be due to: • • Lower affinity for DNJ-NAP Not enough protein (and associated radioactivity) on the gel
CONCLUSIONS
The photolabile DNJ-NAP will only cross-link to its substrate - specific It seems to take 4 - 10 minutes to reach its target and bind Binding is increased after 30 minutes There is no longer binding after 60 minutes Most likely due to cell lysis
FUTURE PERSPECTIVES
Investigation into events between 30 and 60 minutes More detailed analysis of the time-course of the binding reaction – repeats Identify the species to which it is bound
ACKNOWLEDGEMENTS
Dr Terry Butters Dr David Neville, Gabriele Reinkensmeier, Dom Alonzi, and Stephanie Boomkamp Dr Mark Wormald Professor Raymond Dwek