Science Buddies: "Ask an Expert"

I want to alter the amino acid sequence of the HA cleavage proteases that specifically cleave the hemagglutinin of the H5N1 Avian Influenza.

OK. There is more than one protease that does this, and as I think I linked before, which one does it depends upon the site of infection. Two of the major enzymes that do this are trypsin and furin: http://www.ncbi.nlm.nih.gov/pubmed/18235997

Also, what kind of equipment will I need to accomplish my research project ?(my adviser wanted to know)

As in, to actually alter the genes and insert them into embryonic stem cells and then make a transgenic animal? Lots of equipment. I thought the goal was just to design the modifications, not to actually make them.

SandbaggerOne wrote:As in, to actually alter the genes and insert them into embryonic stem cells and then make a transgenic animal? Lots of equipment. I thought the goal was just to design the modifications, not to actually make them.

Yeah, that's what I was supposed to say: altering the design. Do I need any equipment for that?

Nope, just your brain, internet access, and a bunch of reading material.

Ok thanks

What's the difference between Trypsin 1 and Trypsin 2?

They are different enzymes encoded by different genes. Both enzymes have inactive precursors called trypsinogens. These must be cleaved to become active enzymes. In humans trypsin 1 is encoded by the PRSS1 gene and the precursor is called called cationic trypsinogen. Trypsin 2 is encoded by the PRSS2 gene and its inactive precursor is called anionic trypsinogen. They are 90% similar by amino acid sequence. One major difference is that they are stable over different pH ranges.

Does that help?

Can I choose one of them and alter that sequence?

Did you find some information that indicates trypsin 1 and 2 are important for cleaving HA?

SandbaggerOne wrote:

OK. There is more than one protease that does this, and as I think I linked before, which one does it depends upon the site of infection. Two of the major enzymes that do this are trypsin and furin: http://www.ncbi.nlm.nih.gov/pubmed/18235997

.

Thats where I got the idea that trypsin is a protease that cleaves the HA

From what I have read highly pathogenic H5 and H7 viruses (like H5N1), can also be cleaved by several ubiquitous cellular proteases (most notably furin protease). So, targeting trypsin may not be the best strategy. This is frustrating. H5N1 is so dangerous in part because several different enzymes can activate its HA. Trypsin is restricted to the intestines and pancreas, while furin is all over the body. However, studies from mice that completely lack furin (this is called a "knockout") show that they have many abnormalities during enbryonic development, so mutating furin might not work so well either. This is frustrating and I am not sure what to suggest now. I need to think about it.

Can a transmembrane protease serine cleave the hemagglutinin of the H5N1 Avian Influenza Virus? If so, is it possible to modify the sequence of that protease? Also, is trypsin/furin similar to this enzyme, or are there huge differences among them?

Thanks

There are several transmembrane serine proteases that can cleave HA in humans: http://jvi.asm.org/content/84/10/5089.full

TMPRSS2, TMPRSS13, and a variant of TMPRSS2 called MSPL are all candidates.

The transmembrane serine proteases are said to be "trypsin-like". One of these enzymes could be a good target for mutation.

This paper describes mutating TMPRSS2 to create a proteolytically inactive version: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1617224/

"A proteolytically inactive single-point mutant, TMPRSS2(S441A), was generated by substitution of the active-site serine to alanine at position 441 by using site-directed mutagenesis as described by Afar et al. (1). This mutation was shown to abolish enzymatic activity of TMPRSS2 without affecting the level of protein expression in transfected cells (1)."

This was the human version of the protein. If you want to see if you can find that part of the sequence in the chicken gene that might be a good idea: http://www.ncbi.nlm.nih.gov/gene/418528

Is there a serine at position 441 of the chicken TMPRSS2?

Yes, there's actually 3 and possibly even more!

May I ask why I have to mutate the serine at specifically position 441?

May I ask why I have to mutate the serine at specifically position 441?

Because in the paper I linked they found that that was the amino acid responsible for its enzymatic activity.

Hello,

I just wanted to thank you for advising me on my research project and I actually won "Best in Category" for it, and was promoted to the Regional's Science Fair Competition, so thank you! But, I have been wanting to improve on my initial idea on "Redesigning the Amino Acid Sequence of the Transmembrane Protease Serine", and apply it to a more challenging field: Cancer Research.
Here's my idea: since Hepsin is also a transmembrane protease serine found in the human body, and is said to aid substantial growth in prostate cancer cells or any cancer like it, why not mutate the Hepsin gene responsible for prostate cancer cell growth and possibly ablate its function? In addition, I would like to to insert the mutated cDNA into an area of sample tissue and test whether or not my theory worked. My idea is still in process, so some areas do need advice.

Thank you