Logan Research: Glycoprotein Structural Biology
Glycoforms expressed on Thy-1 in Lec1 mammalian cells and in Tn5 insect cells. The glycoforms were determined by using mass spectrometry of peptide fragments.
2D 1H,13C correlation spectrum of recombinant Thy-1 labeled in the carbohdyrates. Tentative assignments are indicated in the figure.
This work is supported by grants from the FSU Research Foundation and the NSF through the National High Magnetic Field Laboratory.
The major challenge facing strutural glycobiologists is the inherent heterogeneity in cabohydrate patterns found even within singly glycosylated glycoproteins. This heterogeneity arises because the glycosylation is not templated, as are the protein or DNA sequence of these molecules. Instead, the specific glycosylation pattern seen at a given site depends on many factors, including the specific pattern of glycosyl transferases found in the Golgi bodies. This heterogeneity must be important for biological function, but it severely complicates crystallization and NMR spectroscopy of these proteins.
We have successfully addressed the glycoform heterogeneity problem by expressing recombinant proteins in Chinese hamster ovary cells that carry mutations in specific glycosyltransferases. For example, expression of Thy-1, a surface glycoprotein containing three different asparagine-linked glycosylations, reduces the number of glycoforms to just four, a very manageable number. These glycoforms are shown in the diagram on the left.
We are currently working to optimize the expression yield and isotope enrichment. Metabolism in mammalian cells is quite different from that in bacteria, and different approaches to isotope enrichment must be developed. We are making some headway, as shown by the high resolution 2D 1H,13C correlation spectra we obtain on this protein.
We are also working to develop novel NMR approaches to characterizing the structure of intact glycoproteins. Some of these methods are for chemical shift assignment, others are for structure determination and characterizing sugar dynamics. This latter project is a collaborative effort with Dr. Jim Prestegard, of the Complex Carbohydrate Center, at the University of Georgia.
For more information, see some of our recent publications:
|Home||Stem Cell Metabolism|