N-glycosylation

[Tilda]

I'm wondering if anyone knows how N-glycosilation works and why some aminoacids get N-glycolysated and some don't? I'm working with the AA-sequence 421-440 for an isoformof human alpha-amylase, if that is to any help. (shown below)

 

Phe Thr Asn Trp Tyr Asp Asn Gly Ser Asn Gln Val Ala Phe Gly Arg Gly Asn Arg Gly

 

Thank you for any help!

/Matilda

[donkey]

are you talking about N-liked glycosylation? Carbohydrates are attached to asparagine residues present in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid (except Pro). I'm not exactly sure why this is off the top of my head but it's possibly due to an enzyme mediated reaction which only recognises specific amino acid sequences. In other words, it may be that some amino acids get glycosylated, while others don't, because enzymes involed have specificity for particular AA sequence. I'd like to stress that that's just my guess so you'd really be better off consulting a text book.

[Tilda]

Yes... N-linked was what I meant You see I'm from sweden and we don't put the "linked-part" in the name Well thank you so much for your help, and your knowledge will be put to great use in my final paper

 

Again thank you

kisses//

 

Matilda

 

ps. GAWD I love this page

[donkey]

hey Matilda,

 

Last night it was late and so I thought I better check my text book to make sure I wasn't talking nonsense.

 

I have checked (Molecular Cell Biology 5th ed., Lodish et al, pp673-675) and it confirms that "the tripeptide sequences Asn-X-Ser and Asn-X-Thr (where X is any amino acid except proline) are substrates for oligosaccharyl transferase, the enzyme that catalyzes this reaction".

 

When it says this reaction it's refering to the transfer of a carbohydrate polymer (i.e. a precursor oligosaccharide - this is a "precursor" because it's later modified in the ER and Golgi apparatus) from a molecule called dolichol to the protein as the new protein is translated and translocated across the membrane of the rough ER. Dolichol is essentially a holding molecule and sits in the membrane holding the carbohydrate waiting for the new protein.

 

So the answer is that oligosaccharyl transferase decides which amino acids get N-glycosylated.

 

Here are a few links that might be helpful:

 

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=oligosaccharyl+transferase+AND+373563%5Buid%5D&rid=mboc4.figgrp.2351

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=oligosaccharyl+transferase+AND+373517%5Buid%5D&rid=mboc4.section.2202#2230

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=oligosaccharyl+transferase+AND+373496%5Buid%5D&rid=mboc4.figgrp.2232

 

edit: that's quite strange, my 2nd post was exactly 12 hours after my first

[Tilda]

Yes i checked The Cell as well, just to confirm =) and you were totally right I wonder, do you possibly know in what direction the protein's pI and the protein's hydrophobic properties are changed when a negative charged group is added to the "sugartree" ( chain)? I'm not sure, but I think the negative group (residues in golgi) would have to be N-acetylneuraminic acid (also called sialic acid or NANA).

 

thanx again //

 

Matilda

 

ps. I acctually found those links like 5 minutes before seeing your post =) mind reading

[donkey]

If you add a negative charge i'd imagine that would stabilise the protein in water (negative charges interact with water) - therefore, i think adding a negative charge would make it more hydrophilic and less hydrophobic.

 

I'm pretty sure that adding a negative charge would also affect the pH at which the protein's charge became zero (i.e. it's pI) but i'm not totally sure whether you'd need to increase the pH (i.e. increase in the pI) or decrease it.

 

umm, hopefully someone else knows...

[donkey]

I *think* it'd lower the pI if you add a negative charge because this page (http://www.rit.edu/~pac8612/webionex/website/html/ione8zvy.html) says "when the pH > pI, a protein has a net negative charge and when the pH < pI, a protein has a net positive charge". So it might be that in general a low pH allows proteins to be more positive and the addition of an extra negative charge would mean that it wouldn't have an overall positive charge until at a slightly lower pH compared to before. Similarly it would also have an overall neutral charge at a lower pH and thus the pI would be lower.

 

So, my guess is that adding a negative group would lower the pI.

[Tilda]

damn, you're good...now this is dangerous... i won't look anything up anymore, I'll just look YOU up and ask ya =)

Well...thanx a bunch..again =)

 

hehe..you've been a GREAT help =)

 

soooo much left to do and so little time. it's mighty hard to find all answers on net, especcially when you have to translate terms into english from Swedish first...hehe and you don't have a master's degree =)

 

so lets just say I'm greatful for all the help I can get =)

 

kisses//

 

Matilda

[Tilda]

Well.. imight as well ask...

 

I can't seem to find which enzymes that are involved in the insulin synthesis. I've used keywords such as "insulin synthesis", "proinsulin" and "mature insulin". Maybe you have an idea on how to solve this easier... don't know if the searching's wrong, maybe I have some term wrong somewhere.

 

Hope I'm not taking too much of your time

 

Might as well ask where you're from/age/male or female =)

[donkey]

hey,

 

I'm happy to help I finsihed my exams for university a couple of weeks ago so have quite a lot of spare time and i'm enough of a geek to enjoy this

 

Lodish et al (5th edition, p725) has some good details.

 

Proteolytic cleavage of proproteins, such as proinsulin, occurs in vesicles after they move away from the trans-Golgi network. The proproteins of most constitutively secreted proteins (e.g., albumin) are cleaved only once at a site C-terminal to a dibasic recognition sequence such as Arg-Arg or Lys-Arg.

 

(check out this link: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.4851)

 

Proteolytic processing of proteins whose secretion is regulated generally entails additional cleavages. In the case of proinsulin, multiple cleavages of the single polypeptide chain yeilds N-terminal B chain [blue bit on the left in the picture above] and C-terminal A chain [blue bit on the right in the picture above] of matrue insulin, which are linked by disulfide bonds, and the central C peptide, which is lost and subsequently degraded.

 

So it looks like PC3 endoprotease, PC2 endoprotease and carboxpeptidase remove all the unwanted amino acids to convert proinsulin to insulin.

 

the online version of an earlier edition of the book (slightly different wording but similar info) is here:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=endoprotease+proinsulin+AND+106720%5Buid%5D&rid=mcb.section.4846#4850

 

hope that helps

 

oh and i'm from the UK (Edinburgh), 22, male and an undergraduate biochemistry student What about you?

[Tilda]

Almost seems like the Lodish version has brought more joy than The Cell Oh well a good spent £100

 

I'm 21 year old female from Sweden (female lol... makes me feel like a chimp or something...oh...right...I almost am )

 

Well I've just (almost... just a tiny bit lft ) ended my first year in biomedicalanylist (I know it has a better title in english, but I've never seemed to remeber it)

 

well... I will check the online version now =)

 

Thanx again...you'll probably hear from me soon again

 

Kisses//

 

Matilda

 

ps. I'm just about to move, so I won't have internet for a while, that's why I've been so desperate to get answers =), but just so you know if I don't answer for a while

[donkey]

have fun moving

[donkey]

hey, I hope you got your assignment all done. I doubt you're still reading this but I'm going to Stockholm later this month and as you're from Sweden I was wondering if you had any suggestions as to what I should see?

[Tilda]

long time no hear =)

 

The assignment went well.-. I passed! probably thanks to you

 

Stockholm ey...hehe nothing to see there=)

well acctually I've only been there once...so sry no othing to tell you accept I'm not that into the city..nothing for me =)

besides us located futher south tend to hmmm... not LOVE the "stockholmers" or whatever you might wanna call them. =)

 

but I hope you have an awsome time and enjoy our country

 

kisses//

 

tilda