Structure of RAP Antibodies (Specifically RAP-5)

Question

[EDIT] - Have just found not one but two papers that address my structure problem. However they concern RAP-1A, so I guess my question is now what is the difference in structure and function of RAP-1A and RAP-5? Does anyone know of X-ray structure analysis being used to examine RAP-5?

---

original question

I'm a University Physics student writing a mock review article on what to me feels like a very 'un-physicsy' antibody - RAP-5.

Although my knowledge of Biology is pretty poor, research is going quite well. I've found a lot of papers from the 80s talking about conducting immunohisto(/cyto)chemistry experiments, most of them finding that RAP-5 can be used to determine whether a cell has the ras in it, so they are able to measure the percentage of cells that are neoplastic (I believe this means cancerous) and contain ras and the percentage of cells which are normal and contain ras. (the ras-gene being a proto-oncogene which on mutation can result in permanently switched on ras proteins (p21?), which results in proliferation of cells and therefore can cause tumors). This is all very nice but on first glance immunohistochemistry doesn't seem to involve a huge amount of physics (for my bio-physics assignment), apart from using an optical microscope.

I was hoping to be able focus a section of my article on the physical techniques involved in determining the structure of RAP-5. Although there seems to be plenty of literature on uses of RAP-5, I am struggling to find anything on the details on why it is able to be used in such experiments. In other words, I presume that its function is to bind on to an epitode specific to the ras protein (amino acids 10 -17 have popped up a few times) but I don't know if there is any imaging one can do to have a look at the structure and conclude 'yes this is why it binds to ras proteins and not to others'. Is there a technique that is likely to have been used to examine the structure of RAP-5? Is it's tertiary structure likely to be 'Y' shaped like other antibodies? Does it differ in structure from RAP1-4? (this book informs me that RAP1 and RAP2 have 60% sequence identity, but most sources seem to leave out RAP3-to-5, some evening telling me that the RAP family consists of RAP1A/B, RAP2A/B/C and no others!).

Also, if RAP-5 is an antibody, does this mean that it is produced in the body and gets involved in the ras protein signal pathway in order to reduce too much ras expression? (am I right in saying the amount of expression is the amount of protein the ras-gene is producing?) or is it only synthetically produced and used in experiments to measure the amount and location of ras proteins?

Also there seems to be little differentiation between the differences in the functions of each RAP. RAP-5 seems to be used quite a bit in experiments involving Ha-ras - but not exclusively. Do the different RAPs bind to different variants of ras? Ha-ras being the one unique to RAP-5.

Answer 1

The confusion that you're facing is because RAP-5 is actually known as RAB5C (GENEID). The ras superfamily (review) is divided into Ras, Rho, Rab, and Rap. But the Rap GTPases are divided only into two categories, RAP1 and RAP2. On the other hand, there are multiple Rab GTPases which include RAB5A and RAB5C.

There are a few crystal structures of both Rab5A and Rab5C in both human and murine forms.

Now, for RAP-5. RAP-5 is a monoclonal murine antibody which has no relationship to the RAP family of proteins. It's probably called RAP because Spandidos and Wilkie weren't thinking clearly when they named their antibodies RAP1-5. In this case, you're probably better off looking at the antibody Y13-259 which is probably called that since its the 259th antibody they tried.

To answer your questions, specifically, very few people have attempted to get a structure since it is an antibody and everyone knows what they look like. Secondly, the antibody is produced in a mouse and added to tissue. And yes, the RAP antibodies bind to different variants of Ras. Also, no one uses RAP-5 since anti-Ras EP1125Y seems to be more popular.

If you still want a crystal structure, I would hunt down the RAP5 patent and look up the peptide sequence. From there, you can look up similar structures in the IMGT database