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RESEARCH FOCUS, BIOGRAPHY, PUBLICATIONS

Research Focus
GLYCOSYLATION: AN ESSENTIAL FUNCTION
The entire surface of cells is coated with sugars built into complex, multi-branched chains. These chains are linked to proteins in the membrane, where they can promote (or sometimes interfere) with cells binding to each other. These sugar chains are first attached to proteins inside the cell where they help proteins “get into shape” for their jobs. As the proteins percolate toward to cell surface, the sugar chains are sculpted for specific needs. This entire process, called glycosylation, recruits a force of more than 500 genes for this job. The Freeze lab works on several facets of glycosylation, all of them with an eye toward therapeutic applications for diseases that impair the functions of these critical genes.

HUMAN GLYCOSYLATION DISORDERS
One of our major areas is a group of inherited diseases is called Congenital Disorders of Glycosylation (CDG). Today we know of defects in 25 of genes compared to just 3 a decade ago. Patients with these diseases have highly variable mental and motor retardation, seizures, failure to grow, hypoglycemia (low blood sugar), clotting and digestion abnormalities, to name just a few. These are rare disorders with less than 1000 known patients worldwide, but it is likely that there are many more patients who remain undiagnosed. Physicians are becoming more aware of CDG and glycosylation in general, and basic scientists continue to discover sugar chains at the helm of many basic metabolic processes. Defective glycosylation is also known to cause several types of muscular dystrophy. Figure 1 shows the explosive growth in the number of different diseases that are caused by defective glycosylation. Figure 2 shows two out of the scores of children with CDG we have worked with.

Figure 1


Our lab is currently focusing on identifying new defects that cause CDG and trying to understand how these defects are translated into the disease presentation. Defects occur in genes that activate and transport sugars, assemble them into glycans, remodel them. Most recently identified are genes that are needed to traffic and distribute the glycosylation machinery within cells. Ongoing collaborations with academic physicians provide a steady flow of new patients for analysis. Since very few laboratories in the United States work on CDG, we are developing new molecular diagnostic methods to handle the increasing number of patients. With the help of generous philanthropic support, we have also recently begun to screen molecular libraries to identify small molecules that can supplement the depleted glycosylation pathways in some of these patients.

PROTEIN-LOSING ENTEROPATHY
Some patients suffer from an often-lethal condition called protein-losing enteropathy (PLE), where blood proteins leak through the intestine, causing massive fluid imbalance. Some CDG patients and children who have had (Fontan) corrective surgery to mend congenital heart defects sometimes develop PLE months to years after their surgery. Its basis, and why PLE strikes only certain children is a mystery. Aided by the Children’s Hearts Fund, the lab has focused on understanding how key molecules and environmental insults interact to drive PLE. We used some of these insights to provide a therapy for one young adult with PLE.

INFLAMMATION AND CANCER
The other major focus in the lab is a new facet of how an unusual sugar chain modification is involved in inflammation including Crohn’s disease, ulcerative colitis, cancer, and arthritis. A monoclonal antibody against this unusual sugar chain modification, called a “carboxylate”, shows encouraging signs as a potential therapy for inflammatory-related diseases. We continue to work on the structure of the sugar chain and its mechanism of action. Only a limited number of proteins carry this “carboxylate” modification and many are known to be involved in cell apoptosis or the development of inflammatory disorders. Recent results show promise for the ability of the antibody to disrupt tumor angiogenesis and growth. These exciting results position this antibody as a candidate for future clinical trials.

NEW DIRECTIONS
A newly funded grant from the California Institute for Regenerative Medicine (CIRM) will explore critical changes in glycosylation that accompany the transition of human embyronic stem cells into neural precursor cells and then differentiate to become neurons, astrocytes, and oligodendrocytes. A collaborative project with Emory University will also explore the basis of abnormal glycosylation seen in patients with galactosemia.

Biography
Dr. Freeze earned his Ph.D. from the University of California at San Diego in 1976. Subsequently he held fellowships in Biology, Medicine and Neurosciences later joined the faculty at the same institution. In 1988 Dr. Freeze was recruited to the Burnham Institute for Medical Research.

Selected Publications
Wu, X., Steet, R., Bohorov, O., Bakker, J., Newell, J., Krieger, M., Spaapen, L., Kornfeld, S., Freeze H.H. Mutation of the COG complex subunit, COG7, causes a lethal congenital disorder. Nature Medicine. 10:518-528, 2004.

Srikrishna G, Turovskaya O, Shaikh R, Newlin R, Foell D, Murch S, Kronenberg M, Freeze HH. Carboxylated glycans mediate colitis through activation of NF-kappa B. J Immunol.175(8):5412-22, 2005.

Freeze, H.H. Genetic Defects in the Human Glycome. Nat Rev Genet, 7(7):537-551, 2006.

DeRossi, C., Bode, L., Eklund, E. A., Zhang, F., Westphal, V., Wang, L., Borowsky, A. D. and Freeze, H. H. Ablation of Mouse Phosphomannose Isomerase (Mpi) Causes Mannose 6-phosphate Accumulation, Toxicity, and Embryonic Lethality. J Biol Chem, 281:5916-5927, 2006.

Bode, L., Murch, S., Freeze, H.H., Heparan sulfate plays a central role in a dynamic in vitro model of protein-losing enteropathy. J Biol Chem. 281(12): 7809-15, 2006.

Kranz, C., Ng, B., Sun, L., Sharma, V., Eklund, E., Miura, Y., Ungar, D., Lupashin, V., Winkel, D., Cipollo, J.F., Costello, C.E., Loh, E., Hong, W. Freeze, H.H. COG8 deficiency causes new Congenital Disorder of Glycosylation type IIh. Hum Mol Genet, 16(7):731-741, 2007.

Bode, L., Salvestrini, C., Park, P., Li, J., Esko, J., Murch, S., Freeze, H.H., Heparan Sulfate and Syndecan-1 are Essential in Maintaining Intestinal Epithelial Barrier Function. J Clin Invest, 2007.

Liem, Y.S., Bode, L., Freeze, H.H., Leebeek, F.W.G., Wilson, J.P.H. Heparin Reverses Protein-Losing Enteropathy in a Patient with Congential Disorder of Glycosylation type Ib. Nat Clin Pract Gastroenterol Hepatol, 2007.

List of Publications via PubMed
(NIH National Library of Medicine)