PRG4/Lubricin glycoform variants associated with endocardial infections and sepsis - PROJECT SUMMARY/ABSTRACT Proteoglycan 4 (PRG4, also known as lubricin) is a mucin-like protein best known for its role as an important lubricant in joint fluid. The O-linked glycans (over 100 per PRG4 polypeptide) contribute to more than 50% of the mass, and are responsible for the lubricating and anti-adhesive properties. Aberrant glycosylation, specifically decreased sialic acid and branched structures, is correlated with impaired rheological properties and osteoarthritis. PRG4 is also expressed in the heart, lungs and liver, and circulates in blood. However, the role in these other tissues is unclear. PRG4 has been reported to dampen inflammatory responses, but has also been linked to pathologies such as aortic valve stenosis and the formation of necrotic hepatic vascular thrombi. Very recently, it has been implicated in the acute phase response to vascular infections, and our own studies have shown elevated plasma levels in animal models of infective endocarditis (IE). We previously used a combination of lectin blotting and mass spectrometry to characterize the O-glycans linked to PRG4 in healthy human plasma, as well as plasma and cardiac valve tissue from rat and rabbit models of IE. We found that the O-glycans on PRG4 from plasma of healthy rats resembled those of healthy humans. However, in addition to increased PRG4 levels, the O-glycan modifications were altered in IE animals (just a single type of trisaccharide structure, rather than the assortment of ~20 different structures seen in healthy animals). We hypothesize that distinct PRG4 glycoforms will likewise be associated with vascular infections in humans, and that these differently glycosylated forms will have different immunomodulatory or mechanical functions. Thus, as a prelude to mechanistic studies, we need to first clearly define the range of plasma PRG4 glycoforms evident in human health and disease. The overall aim of this project is to develop methods for the rapid and high through-put detection of PRG4 glycoforms in minimally processed plasma samples, and then determine the prevalence of aberrantly glycosylated PRG4 in plasma from a cohort of sepsis patients. We will use a cutting-edge glycoproteomics mass spectrometry approach to precisely characterize the differences between PRG4 isolated from our banked samples of rat plasma collected pre- and post-infection. We will also use the banked IE and healthy rat plasma, along with our unique set of streptococcus-derived sialoglycan-binding probes, to develop a high-precision ELISA for determination of PRG4 levels and relative amounts of disease- associated (trisaccharide-modified) glycoforms. We will then determine whether comparably different glycoforms are evident in healthy human and sepsis patient plasma, and assess whether the PRG4 levels or O-glycan variations correlate with sepsis phenotype, or with biological variables such as age, sex, or ethnicity. The results will aid the design of larger mechanistic studies that address how PRG4 impacts vascular repair, coagulopathies and inflammatory responses. Although the focus is on PRG4, the results will be applicable to the detection and characterization of glycoform variants of mucins and mucin-like proteins in general.
