Mutated invariant codons and functionality


#1

I have a question about how we define which BCR sequences are functional. One of the criteria we all seem to use is that the invariant codons (cyst and tryp/phen) are in frame and not mutated. Is anyone aware of studies that have actually measured how frequently this assumption is violated, i.e. how common are sequences with mutated/o.o.f. invariant codons that are actually functional? Or is this a silly question and they’re unlikely to be functional on theoretical grounds?


#2

I’ve observed variants with mutated C/F/W in many of our datasets which are too abundant to be erroneous.

For example referring to our old data (MIGEC paper):

There is a rank 3 clonotype supported by 0.5mln reads out of 37mln (1.5% freq) with no similar variant having higher frequency:

IGHV3-74 - CARDFRAGVPAEYS - IGHJ4

Its hard to believe it is an error as it both has high frequency and we’ve used UMIs to correct errors.

Here is igblast mapping for it:

The variant apper to have some hypermutated subvariants:

0.54% CARDFRAGVPAEFS
1.50% CARDFRAGVPAEYS
0.02% CARDFRAGVPAEYW

So it is likely to be something biologically functional.

Another example for conserved Cys (this has a far lower frequency, 0.02%):

Thus, if a full-length sequence of an IG chain is known I would define functional antibodies as those that do not have stop codons when translated. As for incomplete reads we can rule out cases with a stop codon and a frameshift in CDR3, frameshift indels in V, as well as with V pseudogenes from the functional class.


#3

At the sequence level, the conserved positions are used to generate the alignment. The antibody response selects what ever is functional, so when you find a functionally expressed antibody with mutations in the conserved position in a hybridoma then it must be functional.
If you find such mutations in a mixture of cells during an immune response without recovering the functional antibody, it is difficult to tell. Could be cell that is about to be eliminated…


#4

These are two rather different criteria. I agree with @mikhail.shugay that there are clearly functional sequences with one or both codons mutated (for instance in the VRC26 mAb light chains). But I think in-frame is probably a pretty strict requirement, given that it would affect the relative frames of the constant and variable regions. But, then again, there are those antibodies with the LAIR-1 insertions that Lanzavechia found, so I guess anything’s possible.

I think this is only true if you are sampling from the GC itself, as non-functional cells should never make it into circulation…


#5

In their in vitro evolution study “Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity” (DOI: 10.1073/pnas.170297297) Boder et al noted extensive sets of functional, very high affinity, antibody fragments carrying Leu or Met in place of W118 in the heavy chain. These binders were the results of in vitro affinity evolution but demonstrate that this position is indeed capable of sequence variation and compatible with a relevant protein structure. If that is the case with your sequences is of course a different matter that may require further evaluation at the protein level.


#6

Great, thanks for the thoughts, everyone. That all sounds super sensible.


#7

I have seen non-canonical amino acids replacing the W or F in the J-encoded regions, but I doubt very much that you can have a functional antibody without the two canonical cysteines. These amino acids form a disulfide bond that stabilizes the antigen-binding domain. Without them, the molecule would flop around flaccidly.


#8

As far as I know there is one report on such case http://www.bioc.unizh.ch/plueckthun/pdf/APpub0114.pdf. Anyways I agree that such cases are very rare compared to F/W mutations.


#9

We also have isolated single cells in which the second C (FWR3) codon of IGH-V was mutated. They are rare for sure (<1:1000 in the IgG memory compartment), but we could recombinantly express the two antibodies we found. As the cognate antigen is unknown, I cannot comment on the functionality of these antibodies, just that they are stable.
Mutations in the conserved IGH-J W codon are much more abundant (at least an order of magnitude) and here we have found clear examples in which target specificity was not negatively affected.