Tesar, Devin Brent (2009) Investigations of the mechanisms of receptor-mediated immunoglobulin transport in mammals and birds. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-11122008-131536
The neonatal Fc receptor (FcRn) mediates the passive acquisition of humoral immunity in early pre- or post-natal mammals by transferring maternal IgG to the fetus or suckling newborn. In addition, FcRn serves to extend the serum half-life of IgG in adult mammals by protecting it from a default degradative pathway in vascular endothelial cells. For both of these functions, FcRn binds the Fc domain of IgG with high affinity at the acidic pH (≤6.5) of the intestinal lumen and acidic endosomes, and releases the IgG at the slightly basic pH (~7.4) of blood. While the ability of FcRn to transcytose IgG bidirectionally in polarized cell models has been well documented, the specific mechanism(s) by which endocytosed IgG is sorted from other vesicular cargo and directed through a progression of endosomal compartments ultimately leading to the apical or basolateral membrane are poorly understood.
We wished to develop an in vitro system with which to study the trafficking behavior of rat FcRn in polarized epithelia. I developed such a model system using Madin-Darby Canine Kidney cells stably-transfected with the rat FcRn. The cells bind, endocytose, recycle and bidirectionally transcytose FcRn ligands, faithfully recapitulating the function of FcRn in the neonatal rodent gut. We used these cells to test whether or not the presence of two FcRn binding sites on an Fc ligand are required for transport. The results demonstrated that ligand bivalency is not strictly required for transport but does increase the transcytosis efficiency of the system, an interesting result in light of the fact that FcRn binds and transports a naturally monovalent ligand – serum albumin.
We have used the FcRn-MDCK cells to study trafficking of the receptor in living cells using spinning disk confocal microscopy. For these studies I developed a recombinant Fc ligand containing a tandem dimer of fluorescent proteins on each chain and demonstrated that this ligand is brighter and more photostable than conventional chemically-labeled Fc molecules used in other confocal studies. Our FcRn-MDCK cells have also been used to examine trafficking events at the plasma membrane leading up to exocytosis though use of total internal reflection microscopy (TIRFM).
I also created a model system to study an avian Fc receptor present in yolk sac (FcRY). FcRY has been shown to bind IgY (the avian counterpart of IgG) with the same pH-dependence that FcRn exhibits for IgG. Transcytosis and recycling experiments using polarized rat inner medullary collecting duct (IMCD) cells stably transfected with the FcRY gene demonstrated that this receptor is a true functional equivalent of FcRn despite being structurally distinct from FcRn. This presents a striking example of convergent evolution and demonstrates that certain versatile protein folds can play key roles in more than one functional context within a complex organism.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||FcRn; immunoglobulins; MDCK; polarized epithelia; pssive immunity; receptor trafficking|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||12 September 2008|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||04 Dec 2008|
|Last Modified:||26 Dec 2012 03:09|
- Final Version
See Usage Policy.
Repository Staff Only: item control page