Reading Summaries for Week 2 - Patricia P. Jones

The Invariant Chain of Murine Ia Antigens: Its Glycosylation, Abundance and Subcellular Localization
Ellen Sung and Patricia P. Jones

In this paper, Sung and Jones characterize the glycosylation, abundance, and subcellular localization of Ia antigen-associated invariant chain (Ii). By utilizing the antibiotic tunicamycin (inhibits the addition of N-linked carbohydrates to nascent polypeptides) and 2D polyacrylamide gel electrophoresis, they show that while there are various haplotypes of polymorphic chains, there appears to be only a single precursor molecule for Ii. They also show that Ii is likely to be glycosylated by two N-linked oligosaccharide units. In the second part of the study, they use various membrane extraction and immunoprecipitation procedures to show that Ii does not appear to be associated with any surface Ia antigens nor is it free in the cytosol, yet there does appear to be a pool of Ii chains within the cell that are not bound to Ia antigens. In their conclusions, they suggest the possibility that Ii is associated then only with intracellular polymorphic chains and raise the question: what exactly is the function of Ii?


Identification of an IFN-gamma Responsive Region in an Intron of the Invariant Chain Gene
Zhu A. Cao, Bethany B. Moore, David Quezada, Cheong-hee Chang, Patricia P. Jones

IFN-gamma is known to co-regulate the expression of MHC class II and invariant chain (Ii) in murine macrophages. In this paper, the researchers explore the mechanism by which IFN-gamma upregulates Ii mRNA. By using transient transfections of CAT reporter constructs, they were able to identify a 155-bp region (named Q) within the first intron of the Ii gene that contains an IFN-gamma responsive element. They further confirmed this by testing reporter constructs containing various mutations in gene promoter boxes--any mutation in the Q fragment eliminated IFN-gamma responsiveness. However, the Ii promoter region and upstream enhancer most likely work synergistically with fragment Q to mediate IFN-gamma induction of Ii. Sequence analysis of Q revealed homologous regions to motifs in MHC class II gene promoters and the Ii enhancer. It is likely then that expression of these various proteins is regulated by similar mechanisms.


Questions asked in class plus some:
If MHC haplotypes preferentially recognize and bind certain kinds of peptides that contain similar motifs, then how is it that all MHC haplotypes form a complex with the same invariant chain?

Why would nature put such stock in one single version of the invariant chain? What if there is a mutation? Have any mutations been reported/any research done involving mutated invariant chains?

How exactly does 2D-PAGE work?

How exactly does an intronic enhancer work?

What is the difference between N-linked and O-linked glycosylation (aside from the obvious, i.e. what physiological differences might occur)? Why choose to study N-linked instead of O-linked?

Feel free to add more in the comments!