INVESTIGATION OF HEPARAN SULFATE 3-O-SULFOTRANSFERASE-1 ACTIVITY AND INHIBITION VIA TANDEM MASS SPECTROMETRY

Heparan sulfate (HS) is a structurally complex and highly sulfated glycan whose biological functions are dictated by precise sulfation patterns. Among these modifications, 3‑O‑sulfation, introduced by heparan sulfate 3‑O‑sulfotransferase‑1 (3OST‑1), is extremely rare yet functionally critical. This modification contributes to essential physiological and pathological processes, including anticoagulation, viral recognition, Alzheimer’s disease pathology, and cancer progression. Despite its importance, the direct quantification of 3OST‑1 activity in biological systems has remained a major challenge, largely due to the lack of sensitive and structurally specific analytical methods.This dissertation presents the development of a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay that overcomes these limitations by integrating a structurally defined HS hexasaccharide substrate, 13C‑labeled internal standards, exhaustive heparinase digestion, and AMAC derivatization. The resulting method enables nanogram‑level detection of the diagnostic 3‑O‑sulfated trisaccharide product and exhibits a 100‑fold improvement in sensitivity compared to traditional HPLC‑UV detection. Using this platform, endogenous 3OST‑1 activity was quantitatively measured for the first time in HCT‑116 cells and their conditioned media, revealing both intracellular and previously unrecognized extracellular enzyme activity.Leveraging this high‑sensitivity assay, the dissertation further characterizes 3OST‑1 E86Q, a rationally designed dominant‑negative (DN) mutant. Structural rationale and binding studies indicate that E86Q retains affinity for both substrate and PAPS donor but eliminates catalytic function. Biochemical titration assays demonstrate that E86Q suppresses wild‑type 3OST‑1 activity by >80% in vitro, and cellular studies confirm that E86Q significantly reduces 3‑O‑sulfated HS products without altering overall HS abundance or upstream sulfation events. These findings validate E86Q as a high-fidelity DN inhibitor that selectively interferes with the terminal 3‑O‑sulfation step.Together, the LC-MS/MS assay and the E86Q dominant‑negative strategy establish a new experimental framework for probing the enzymatic, structural, and biological roles of 3OST‑1. This work provides the first quantitative tool for assessing 3OST‑1‑like activity in complex biological matrices and offers new opportunities for biomarker development, mechanistic studies of HS fine structure, and therapeutic targeting of HS‑related pathways in disease.