Tolbert Lab: Glycoprotein Research

Glycoprotein Research

The erythrocyte glycocalyx is formed of glycoproteins and glycolipids and is up to 1400 Å thick. Voet & Voet, Biochemistry 3rd edition.

Glycoproteins play many important roles in biology and medicine, covering the surfaces of cells, mediating cell-cell recognition events, and forming a large fraction of FDA approved protein therapeutics. Unfortunately glycoproteins are also very difficult biomolecules to work with due to the nature of their biosynthesis. We are developing methods to produce homogeneous glycoproteins for biochemical and structural studies of protein glycosylation.

During transcription of DNA into RNA and translation of mRNAs into proteins, the sequence of nucleic acids encode for the formation of a single homogeneous protein product (excepting splicing variants). Protein glycosylation, on the other hand, is not a homogeneous process, and will convert a single protein into several glycosylated versions of that protein. The formation of the multiple glycosylated variants (glycoforms) of a given glycoprotein is referred to as microheterogeneity, and is a major difficulty in working with glycoproteins.

Microheterogeneity severely complicates the study of glycoproteins. Different glycoforms of a given glycoprotein can have different biological activities, molecular weights, isoelectric points, and stabilities. These different physical behaviors can make purification of significant quantities of a single glycoform from a microheterogenous glycoprotein mixture a very difficult task, and complicates the study of the function of glycosylation in glycoproteins. Glycosylated proteins such as erythropoietin (EPO) can have a multitude of glycoforms which can differ according to the type of cell the glycoprotein is produced in.

Isoelectric focusing of EPO samples. (a) normal urinary EPO, (b) recombinant EPO, and (c) EPO recovered from the urine of a subject after recombinant EPO injections (Chem. Soc. Rev., 2004, 33, 1-13)

Utilizing yeast to make homogeneous glycoproteins. Yeast are eukaryotic organisms that produce glycoproteins in a similar manner to humans. Though the type of glycosylation on yeast glycoproteins differs greatly from human glycoproteins, yeasts are good organisms for protein production and genetic manipulation of glycoprotein biosynthetic pathways.

Yeast vs. Human glycosylation. The initial stages of yeast and human N-linked glycoprotein biosynthesis are very similar, sharing a common high mannose intermediate (Man₈GlcNAc₂), but the later stages diverge greatly. Humans trim back mannose residues and add a variety of different sugars including galactose, N-acetylglucosamine, and sialic acid. Yeast tend to build larger oligosaccharides using mannose as a building block.

Studies of glycoprotein biosynthesis by Chiba et. al. (J. Biol. Chem., 1998, 273, 26298-26304) have demonstrated that human-like high-mannose oligosaccharides can be produced in glycoprocessing deficient yeast strains.

We are currently developing chemoenzymatic and biosynthetic techniques that allow the production of homogeneous glycoproteins and oligosaccharides. Ultimately, homogeneous glycoproteins produced in glycoprocessing deficient yeast will be utilized in biochemical and structural studies of the function of protein glycosylation.