Glycan analysis of tissues from a ubiquitin ligase knockout mouse
CFG Member?
No
Request Details
Requester First Name:
Kevin
Requester Last Name:
Glenn
Head of Lab. First Name:
Henry
Head of Lab. Last Name:
Paulson
Assigned First Name:
Henry
Assigned Last Name:
Paulson
Requester Email:
kevin-glenn@uiowa.edu
Request Date:
10/13/2005
Institution:
University of Iowa
Shipping Address:
Attn: Kevin Glenn
Henry Paulson Laboratory
240A EMRB
University of Iowa
Iowa City, Iowa 52245
Comments:
I and my mentor Dr. Henry Paulson, are currently collaborating with your core H to study the glycan affinity of a unique set of ubiquitin ligases that recognize misfolded glycoproteins. I would also like to collaborate with core C to perform glycan profiling on mice that have these important ubiquitin ligases knocked out. I currently have one knockout in hand and I am making a second. I would like to submit the tissue of the first knockout to core C.
CFG Core:
C2
Amount Requested:
Experiment to be conducted:
I am a Postdoctoral Fellow in Internal Medicine working in Dr. Henry Paulson’s lab (http://www.medicine.uiowa.edu/paulson/), where we are investigating a novel family of ubiquitin ligases that have a unique ability to recognize misfolded glycoproteins. We have recently made a knockout mouse for the first identified member of this five gene/protein family, FBG1 (a brain enriched protein also known as Fbx2), and are characterizing the CNS phenotype. In collaboration with Core H, we have already shown that FBG1 has high affinity for the high-mannose glycan moiety of glycoproteins. We are now in a unique position to examine the effect that knocking out one of the few known glycoprotein-specific ubiquitin ligases has on glycoprotein homeostasis.
We are committed to the study of glycoprotein processing by ubiquitin ligases, and we recently cloned all human family members (FBGs 1-5), created antibodies to each encoded protein and have begun to assess their different glycan binding affinities. We currently have a mouse knockout of FBG1 and are making mouse knockouts of the other family members as well.
These FBG proteins possess structural homology to the galectin family of proteins (1). The five FBGs, which only exist in complex metazoans, show a high degree of similarity to one another yet are differentially expressed in various tissues. This fact, together with our preliminary work showing differential glycan binding, leads us to hypothesize that individual members of this class of ubiquitin ligases will each bind a unique set glycoproteins.
In fact we believe that a subset of protein from this family (FBGs 1, 2 and 5) are involved in processing misfolded glycoproteins from the ER. Two of the five, FBG1 and FBG2, were previously known to bind high-mannose N-linked glycans, a sugar motif found primarily on nascent glycoproteins in the ER (2,3). In co-immunoprecipitation studies employing the lectin ConA as a probe, we have demonstrated high-mannose glycoprotein binding for FBGs1, 2, and 5. Through collaboration with your core H group, led by Dr. Rick Alvarez, we have extended these studies and obtained individual glycan affinities for FBG1, which confirm our earlier work.
Significantly, proteins in this family of ubiquitin ligases are the only known ligases to specifically recognize and target glycoproteins for proteasomal degradation. Hence knocking out these ligases in a mouse model should have a profound impact on the on the organism’s ability to process glycoproteins. By examining the glycoprotein profile of these knockout mice we might gain insight into the function on these FBG members in glycoprotein processing in both normal and diseased states.
We request a collaboration with the Core C group to perform mouse tissue glycan profiling of our FBG1 knockout, and others as they become available.
1. Mizushima, T., T. Hirao, et al. (2004). "Structural basis of sugar-recognizing ubiquitin ligase." Nature Structural & Molecular Biology 11(4): 365-70.
2. Yoshida, Y., T. Chiba, et al. (2002). "E3 ubiquitin ligase that recognizes sugar chains." Nature. 418(6896): 438-42.
3. Yoshida, Y., F. Tokunaga, et al. (2003). "Fbs2 Is a New Member of the E3 Ubiquitin Ligase Family That Recognizes Sugar Chains." J. Biol. Chem. 278(44): 43877-43884.
Within Scope of Consortium:
Y
Specific aims being addressed:
* 1. Define the specificity and affinity for carbohydrate ligands
* 7. Determine whether regulation of glycosylation modulates GBP function
Comments By Investigator
The request is closed, the data it produced will not generate additional data for this data base. This work will be incorporated into a future publication concerning the FBG1 knockout mouse.