ApoB Structure and Function

#1

Behe’s book as resulted in a lot of attention being focused onto one gene: ApoB. I thought it might be useful for the scholars here at PS to do some background work on what ApoB does, what effects mutations may have, and possibly what effects the specific changes in the polar bear ApoB gene may have.

I found a useful review article:

It would appear that ApoB has two main functions: getting cholesterol into the blood stream and getting it out of the blood stream. Therefore, high or low levels of lipids in the blood stream can be affected by lipid production and lipid clearance. I have only briefly skimmed the abstract of the paper and thought it would be a great topic for the scholar forum.

Interestingly, ApoB is found in two isoforms through post-transcriptional modification: apoB-48 and apoB-100. It would appear that the smaller isoform is responsible for transferring dietary lipids into the blood stream. This may hold a key for understanding possible active sites for lipid production and lipid clearance, and the possible effects of certain mutations in those domains.

For now, I don’t think we need to specifically address Behe’s larger arguments. Rather, we could narrowly focus on understanding the role of ApoB in lipid metabolism.

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Part 2 of Polar Bear Seminar
A Scholarly discussion of ApoB
(Curtis Henderson) #2

One of the really interesting things about the isoforms is that the smaller apoB-48 is a product of specific RNA editing that introduces a premature stop codon. It appears that mutations affecting the C-terminal portion of the protein would be much more of an issue for the apoB-100 vs the apoB-48, resulting in a possible effect on LDL uptake, without significant effect on chylomicron production. I haven’t done any investigating yet, but it would be interesting to see where the mutations have occurred in the polar bear version of the gene.

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#3

I found that interesting as well. I had to go back and change posttranslational to posttranscriptional in the opening post because I had assumed proteolytic cleavage was responsible for the isoforms. Goes to show what assuming things gets you.

That’s my read as well. Here is the figure from the polar bear paper:

For reference, human apoB-100 is 4,536 amino acids long while apoB-48 is comprised of the first 2,152 amino acids (i.e. the N-terminal portion). The mutations listed in the figure are only those expected to affect protein structure according to Polyphen.

At first glance, there could be changes in both functions: transfer of dietary lipids into the blood stream and clearance of serum lipids into cells. The greatest danger that I see is assuming every mutation was selected for. If I am getting my population genetics right, some neutral mutations could have hitchhiked along with the beneficial mutations.

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(S. Joshua Swamidass) #4

That seems correct. It is also possible they are epistatically linked: more than one of them together is required for the function.

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#5

As an aside . . .

If memory serves, @Mercer is familiar with the molecular biology of cardiomyopathies, so there might be something worth pursuing in this section of the polar bear paper:

(Curtis Henderson) #6

Yes, this was interesting, too. I haven’t read up thoroughly, but I don’t recall the cardiac function genes coming up earlier. Presumably, cardiac function would need to increase for the environment and the greater need for swimming efficiency. Behe would have a hard time arguing that these mutations were “breaking” their genes.

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(John Mercer) #7

I doubt that any of them are LOF.

It looks like the authors didn’t bother to pursue any of them beyond looking them up in OMIM, given the laundry-list style they used.

What’s far more important IMO is functional assays on the polar bear APOB alleles.

#8

I don’t know if this was mentioned in previous threads, but polar bear cholesterol levels are extremely high:

This would seem to indicate that apoB-48 is fully functional since that portion of the protein is responsible for transporting lipids from the intestine into the blood stream and lymph. If apoB-48 had lost function then we would expect to see hypocholesteremia.

But what about hypercholesterolemia (i.e. high cholesterol in blood stream)? The review article lists four mutations in humans that interfere with the LDL binding domain: R3500Q, R3500W, R3480W, and R3531C. After a little digging over at Uniprot and a quick alignment, here is the region of the human and polar bear proteins covering those arginines:

Bear   3361  DIKGSVFSREYSGTIASEASTYLNSKSTRSSVKLQGASKVDDIWNLEVKENFAGEATLQR  3420
             D+KGSV SREYSGTIASEA+TYLNSKSTRSSVKLQG SK+DDIWNLEVKENFAGEATLQR
Human  3499  DVKGSVLSREYSGTIASEANTYLNSKSTRSSVKLQGTSKIDDIWNLEVKENFAGEATLQR  3558

Query  3421  IYGIWEHNTKNNLQLEGFFSSSGEHTSKATLELSPWKMSALIQVHASQPSSLLEINYLGQ  3480
             IY +WEH+TKN+LQLEG F ++GEHTSKATLELSPW+MSAL+QVHASQPSS  +   LGQ
Sbjct  3559  IYSLWEHSTKNHLQLEGLFFTNGEHTSKATLELSPWQMSALVQVHASQPSSFHDFPDLGQ  3618

The numbering is a bit off, so R3500 is the R at RSSVKL. In fact, all three R’s in that top row are involved in the mutations related to hypercholesterolemia in humans, and all three R’s are not mutated in the polar bear gene. Therefore, the high levels of cholesterol in the polar bear are not due to known mutations in humans.

At first glance, there is no evidence as of yet that any function has been lost in polar bear apoB, but further investigation is needed. Two of the remaining questions are why polar bear cholesterol levels are so high, and how are they able to tolerate these high levels without suffering from atherosclerosis.

BIG EDIT!!
There is also an H3543Y mutation in humans related to high cholesterol, and the same H is an N in polar bears. This definitely deserves more investigation. I somehow missed this on the first go through.

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#9

Since the last post focused on the human and polar bear comparison, it is probably important to compare the same region between brown bears and polar bears:

Polar  3364  GSVFSREYSGTIASEASTYLNSKSTRSSVKLQGASKVDDIWNLEVKENFAGEATLQRIYG  3423
             GSVFSREYSGTIASEASTYLNSKSTRSSVKLQGASKVDDIWNLEVKENFAGEATLQRIYG
Brown  3372  GSVFSREYSGTIASEASTYLNSKSTRSSVKLQGASKVDDIWNLEVKENFAGEATLQRIYG  3431

Query  3424  IWEHNTKNNLQLEGFFSSSGEHTSKATLELSPWKMSALIQVHASQPSSLLEINYLGQEVS  3483
             IWEHNTKNNLQLEGFFSSSGEHTSKATLELSPWKMSALIQVHASQPSSLLEINYLGQEVS
Sbjct  3432  IWEHNTKNNLQLEGFFSSSGEHTSKATLELSPWKMSALIQVHASQPSSLLEINYLGQEVS  3491

This is the same region where several mutations in humans are responsible for high cholesterol. In the case of the polar and brown bear, they have identical amino acid sequences in this region. Therefore, if there were any “damaging” mutations then they originated in the ancestor of both brown bears and polar bears. This would include the possible mutation at the orthologous H3543 position from the human protein.

(John Mercer) #10

It may be as simple as not living long enough.

How can it be “that mutation” if it’s different?

#11

That doesn’t seem to be the case:

It is interesting that humans don’t tolerate higher levels of cholesterol in the same way herbivores don’t.

That is probably the result of multiple edits. Will fix.

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(John Mercer) #12

Thanks. Even more simple, then!

Still, the way forward is to assay the function of the mutant ApoEs.

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#13

Falling further down the rabbit hole:

So what are the cholesterol levels in polar bears, and in brown bears for that matter?

Brown bears: 7.89 ± 1.96 mmol/L (11.08 ± 1.04 mmol/L while hibernating)
ref

Polar bears: females (median): 8.58 ± 1.31 mmol/L, males: 6.61 ± 2.06 mmol/L
ref

For reference, it is suggested that humans have less than 200 mg/dL cholesterol which is 5.5 mmol/L. Hibernating brown bears have twice that level, while active brown and polar bears are about 50% above that (i.e. 300 mg/dL).

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(Mikkel R.) #14

On a related note, how does dietary fat content affect cholesterol levels? Presumably if you eat lots of fat, your cholesterol levels should go up because that’s one of the functions of cholesterol, to transport dietary fatty acids?

(Andy Walsh) #15

Well, I’ve certainly learned a lot from this thread about lipid metabolism.

Given that carnivores tolerate higher levels of circulating cholesterol, are the human and mouse studies of APOB mutations even relevant to brown and polar bears?

Also, I got curious about the diet of brown bears and polar bears, and found this paper:

In addition to the research on brown bear diet selection, it includes some discussion of the literature on brown bear and polar bear diets, as well as the diets of other bears and other carnivores. They note that brown bears themselves have a high lipid diet relative to other carnivores. They also note that the literature on polar bear diet is not clear cut. So perhaps the diets and lipid metabolisms of brown bears and polar bears are not so drastically different?

Of course, we are still left with the observation that APOB shows evidence of positive selection in polar bears. Perhaps it is still related to diet. My sense from the brown bear paper is that they may choose a high lipid diet possibly similar to polar bears’ (a) if it is available and (b) depending on one’s reading of the polar bear literature. Still, in the wild there may still be meaningful differences if polar bears only have high lipid options while brown bears have a more varied diet with lipid content changing based on availability and timing relative to hibernation. But I’m also wondering if hibernating/not hibernating is a bigger difference than diet between brown bears and polar bears, and if that has anything to do with the APOB differences given that circulating cholesterol is higher in hibernating brown bears than in polar bears.

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#16

I think the studies on mice and humans are of limited value. The elevated levels of circulating cholesterol seem to be a shared feature in bears and aren’t a result of polar bear specific mutations.

As you mention, there is a strong signal of selection in polar bear ApoB. Given the comparable levels of circulating cholesterol in brown and polar bears, I don’t think it has anything to do with the prevention of atherosclerosis. Like you, I am leaning towards adaptations in lipid processing, perhaps related to the higher levels of mammalian fat in the polar bear diet. I could be wrong, but I think high lipid diets in brown bears are usually from fish fat. If they are hunting down mammalian prey they are going to be species like deer which are low in fat.

The polar bear study didn’t measure circulating cholesterol in hibernating polar bears. For the moment, I think it would be safest to assume that the levels are similar in hibernating polar and brown bears.

As an aside, I certainly wouldn’t volunteer to be the person hunting down hibernating polar bears and drawing their blood.

(Andy Walsh) #17

That makes sense. In the paper I linked above, the brown bears had options of salmon oil, and beef fat and pork fat as lipid sources, but that was in a controlled setting.

Especially since polar bears don’t hibernate like brown bears. They don’t become dormant, although they do go through metabolism changes.

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(Mikkel R.) #18

I’ve been thinking the same thing. If the polar bear diet, because nothing else is available, has much more fat in it compared to the average brown bear diet, it would make sense that everything related to fat processing is under selection to improve.

In the arctic every fraction of a calorie potentially matters, probably more-so than it does for brown bears. That implies selection to improve fat uptake into blood through the gastrointestinal wall(let nothing go to waste), which probably partially involves improving fat transport through the bloodstream, improve fat storage (get it into cells), and improve the capacity to turn it into energy to power metabolism and muscular activity. Such adaptation could easily go beyond mutations in ApoB ofc.

I could be wrong, but I think high lipid diets in brown bears are usually from fish fat. If they are hunting down mammalian prey they are going to be species like deer which are low in fat.

It might be worth noting that seal and whale fatty acid constituents is actually significantly more like the fatty acid contents found in fish, and quite unlike the fatty acid distribution found in terrestrial mammals like pork and beef meats.

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(Andy Walsh) #19

I haven’t had a chance to go through it all, but here is another paper (also from 2014) comparing the genomes of polar bears and brown bears.

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#20

I was looking to see if there was any findings related to fatty acid metabolism. This is the best I could find:

So there is some evidence of positive adaptation related to lipids. However, big data papers like this one often create more questions than answers. Another massive rabbit hole would be to look at NO regulation of lipid metabolism in the liver and possible contributions from ApoB. As usual, you go looking for a simple answer and find 10 complex questions.

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