Heparan sulfate co-polymerase function and defects in disease

Proteoglycans harboring heparan sulfate (HS) chains are widely found on cell surfaces and in extracellular matrices where they interact with growth factors, receptors, morphogens, and extracellular matrix components and play critical roles in processes such as cell survival, division, migration, differentiation, pathogen binding,

and cancer development. HS biosynthesis is a complex process involving initial formation of a linker glycan on proteoglycan core proteins, priming and extension of the HS chains’ polymer backbone, and subsequent modification by epimerization, and N- and O-sulfation. The extension of the polymer backbone is a crucial step

in HS synthesis, facilitated by the EXT1-EXT2 heterodimeric co-polymerase complex. Homozygous defects in either of these proteins cause embryonic lethality, and heterozygous loss of function results in hereditary multiple exostoses (HME) characterized by clinical features in bone, cartilage, ligaments, and subepithelial layers,

including the formation of benign cartilage-capped tumors. We recently solved the structure of the human EXT1- 2 heterodimeric co-polymerase in complex with substrate analogs and performed complementary enzymatic studies on both wild type and mutant enzymes to gain insights into HS chain synthesis. EXT1 and EXT2 form

an obligate heterocomplex of the two homologous proteins. Each protein contains two separate predicted catalytic domains, yet only one of the two domains are active in each protein suggesting that the monomers share catalytic functions. We also discovered an interaction between EXT1-2 and the HS priming enzyme

EXTL3. These studies raise important new questions about how HS synthesis is achieved in vivo and the nature and pathology of HME mutations. The prior accomplishments set the stage for our present aims. Aim 1 will test hypotheses regarding EXT1-2 active site residues by probing their roles in substrate recognition and catalysis

and the roles of divergent residues in the inactive homologous domains that restrict catalysis. Aim 2 will test hypotheses on how HME mutations lead to EXT1-2 loss of function and how haploinsufficiency leads to HS loss and pathology. Aim 3 will test hypotheses that interactions with proteoglycan core proteins and upstream

biosynthetic enzymes contribute to efficient HS synthesis. Thus, this research will advance our understanding of HS biology and its roles in health and disease. Unraveling the mechanisms governing HS backbone synthesis will shed light on the molecular basis of HS-mediated cellular processes and pave the way for future development

of targeted interventions.