Determining the Role of O-fucose Modifications in Stabilin-2 Receptor Expression and Function in Human Cells

Aiah Nour Author

08/03/2020 Added

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[00:00:01.220]Hello everybody, my name is Aiah Nour,
[00:00:03.490]and I'm a senior biochemistry major
[00:00:05.810]at the University of Nebraska-Lincoln.
[00:00:08.680]I worked under Dr. Edward Harris
[00:00:10.620]at the Department of Biochemistry
[00:00:12.470]on determining the role of fucose modifications
[00:00:15.910]in the, on stabilin-2 receptor expression and function.
[00:00:20.090]Our enzyme of interest is protein O-fucosyltransferase 1,
[00:00:23.930]or POFUT-1. This enzyme is a glycosoltransferase,
[00:00:27.710]which means that add sugar, fucose specifically
[00:00:30.000]to certain substrates
[00:00:31.160]as a form of post-translational modification.
[00:00:34.290]In order for fucose to be added to these substrates
[00:00:36.900]the enzyme needs to be able to recognize
[00:00:38.910]a consensus sequence on the protein domains,
[00:00:41.510]known as epidermal growth factor domains,
[00:00:43.460]or EGFs, on the protein.
[00:00:45.670]The consensus sequence is as follows,
[00:00:48.000]the second cysteines residue
[00:00:49.500]followed by a series of four amino acids,
[00:00:52.110]which can be any four amino acids,
[00:00:54.210]where the fifth amino acid is either serine or threonine,
[00:00:57.500]followed by the third cysteine residue.
[00:01:00.810]So the reason why the fifth amino acid has to be either
[00:01:03.870]serine or threonine is because
[00:01:05.470]POFUT-1 adds fucose to the oxygen of the hydroxyl group
[00:01:08.690]of the amino acid.
[00:01:10.270]And because serine and threonine are the two amino acids,
[00:01:13.760]who in their basic fundamental structure as amino acids,
[00:01:17.100]have this hydroxyl group,
[00:01:18.370]it has to be either those two amino acids.
[00:01:21.910]There are over 100 human proteins
[00:01:23.680]that have this consensus sequence that can, therefore,
[00:01:26.460]hypothetically be modified by POFUT-1.
[00:01:29.220]Stabilin-2 is one of these proteins.
[00:01:31.290]Stabilin-2 is a transmembrane receptor
[00:01:33.280]that is mostly involved in hyaluronic acid clearance
[00:01:35.760]from the extracellular matrix of a cell.
[00:01:38.300]Other functionalities of Stabilin-2 include
[00:01:40.543]hypoxic cell clearance,
[00:01:41.750]which is the removal of dead cell material in cell fusion.
[00:01:46.620]Stabilin-2 has 20 EGF domains,
[00:01:48.820]six of which require this specific consensus sequence
[00:01:51.450]to be modified by POFUT-1.
[00:01:53.660]So we're determining whether or not
[00:01:55.220]stabilin-2 is a substrate of POFUT-1
[00:01:57.660]and if it becomes modified by this enzyme.
[00:02:00.110]So our overarching research questions are,
[00:02:02.487]"Are any of the six EGF domains on stabilin-2
[00:02:05.460]O-fucosylated by POFUT-1
[00:02:07.480]and what are their- what is the relationship
[00:02:09.330]between these two proteins?"
[00:02:11.250]So in order to examine this,
[00:02:12.810]we had to run expression and function tests.
[00:02:15.600]We first tested stabilin-2 expression in
[00:02:17.870]controlled POFUT-1 cells and POFUT-1 knockout cells.
[00:02:21.320]We used HAEK293 cells
[00:02:24.620]and had one cell line undergo CRISPR-Cas9 genomic editing
[00:02:28.390]to inactivate the POFUT-1 gene.
[00:02:31.170]And both cell lines were grown and cultured
[00:02:33.480]in the same environment.
[00:02:35.050]We ran our samples on five percent SDS-PAGE gels
[00:02:38.210]and ran western blots to examine protein expression.
[00:02:41.340]As we can see here, we see ample protein expression
[00:02:44.550]in both the control and knockout cell lines.
[00:02:47.510]This is just a ladder marker for protein mass.
[00:02:53.430]And we even see protein differentiation in terms of isoform.
[00:02:57.350]Here we have the large stabilin-2 isoform of just 315 here,
[00:03:00.880]and the small stabilin-2 isoform, which is 190 here.
[00:03:05.190]So, immediately we can conclude that stabilin-2 expression
[00:03:09.550]is not necessarily reliant on POFUT-1 presence
[00:03:12.070]in O-fucosylation.
[00:03:13.800]So the next question we have to ask
[00:03:15.650]was whether or not it's protein function was the same.
[00:03:19.250]So we took it to important functionality as a
[00:03:21.230]hyaluronic acid clearance receptor
[00:03:22.940]and did functionality tests
[00:03:24.170]to determine its ability to uptake hyaluronic acid,
[00:03:27.030]in both the control and cell lines.
[00:03:29.310]We exposed both of the cell lines to
[00:03:31.410]radiolabeled hyaluronic acid,
[00:03:32.960]and these are the same cells
[00:03:34.030]that underwent the expression tests,
[00:03:36.950]and examined clearance rate after four hours.
[00:03:39.830]So, we can see here from these two graphs,
[00:03:42.090]these are essentially the same tests.
[00:03:44.030]This is a kinetic test, Figure 2,
[00:03:46.470]that was done multiple times and graphed on a single graph.
[00:03:49.610]And then, Figure 3 here
[00:03:51.350]is a single-time test between the control and knockout.
[00:03:54.660]So we can see that there are slight differences
[00:03:57.100]in clearance rate,
[00:03:58.600]we see in Figure 2 that the knockout did slightly better
[00:04:01.600]and then we see in Figure 3 the control did slightly better.
[00:04:04.990]So we can see from these graphs that although
[00:04:07.180]there are slight differences in clearance rates,
[00:04:10.060]they aren't drastic enough to be attributed to
[00:04:12.720]O-fucosylation or lack of O-fucosylation.
[00:04:15.870]These results were sent to Dr. Bob Haltiwanger in Georgia,
[00:04:19.600]who is the Chief Research on POFUT-1 and O-fucosylation.
[00:04:23.670]And he agreed that these results are negligible
[00:04:25.770]and we cannot conclude from the ligand binding
[00:04:28.460]of hyaluronic acid to stabilin-2
[00:04:30.670]was altered because of O-fucosylation.
[00:04:34.990]So from our experiments,
[00:04:36.100]we can conclude that stabilin-2 expression and function,
[00:04:38.740]are not affected by POFUT-1 presence or O-fucosylation.
[00:04:42.360]The western blot image shows clear expression
[00:04:44.500]in both cell lines
[00:04:45.540]and we can see from the hyaluronic acid function graphs
[00:04:48.370]that differences are negligible.
[00:04:50.320]So from here, we must determine whether or not
[00:04:52.430]any of the six EGF domains on stabilin-2
[00:04:55.080]are O-fucosylated or not.
[00:04:57.070]This requires protein purification,
[00:04:59.880]which will be our next step in the lab.
[00:05:01.640]So once the protein is purified, in our lab,
[00:05:03.570]we'll send it to the Complex Carbohydrate Resource Center
[00:05:06.640]in Athens, Georgia,
[00:05:07.680]to perform molecular analyses on the individual EGF domains.
[00:05:12.320]And the reason we're doing the individual molecular analysis
[00:05:15.510]of the EGF domains is to determine whether
[00:05:19.170]any of the six EGF domains are O-fucosylated by POFUT-1,
[00:05:23.010]and this is important because if we find out
[00:05:25.520]that the EGF domains are O-fucosylated,
[00:05:28.150]that means that POFUT-1 recognized stabilin-2,
[00:05:32.090]modifies it,
[00:05:32.930]but it's just not as important as we see in other receptors,
[00:05:36.580]such as NOTCH receptors, that are modified by POFUT-1.
[00:05:39.770]And if the EFG domains aren't O-fucosylated
[00:05:43.080]that means that POFUT-1 doesn't recognize
[00:05:45.170]stabilin-2 as a substrate.
[00:05:46.870]And that raises questions into,
[00:05:49.460]there must be some other secondary identifiable factor
[00:05:53.780]for an enzyme to recognize a substrate
[00:05:56.190]other than just the consensus sequence
[00:05:57.840]that we see in previous proteins that have been proven
[00:06:00.630]to be O-fucosylated by POFUT-1.
[00:06:02.960]So the reason for the protein purification
[00:06:04.930]is to gain more insight into
[00:06:06.750]the method of O-fucosylation itself
[00:06:08.620]and its relation with different substrates.
[00:06:12.170]So just some acknowledgements,
[00:06:14.230]obviously thank you Dr. Edward Harris
[00:06:16.150]for allowing me to work with him on this research,
[00:06:20.520]the NIH grant that funds Dr. Harris's lab,
[00:06:23.110]and then the McNairs Scholars Program,
[00:06:24.910]which has allowed me to take part in the
[00:06:28.220]McNair Summer Research Experience. Thank you.

Determining the Role of O-fucose Modifications in Stabilin-2 Receptor Expression and Function in Human Cells

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Determining the Role of O-fucose Modifications in Stabilin-2 Receptor Expression and Function in Human Cells

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