A Novel Fluorescent Immuno-Lectin Assay to Identify Osteoarthritis Associated Glycoforms of Lubricin

Abstract

Lubricin or proteoglycan-4 (PRG-4) is a mucinous glycoprotein lubricating the cartilage and maintaining normal tissue function and cell-homeostasis. AlteredO-glycoforms of lubricin has been found in osteoarthritis (OA) synovial fluid (SF). Here, we utilized a Fluorescent Immuno-Lectin Assay (FILA) to measure the levels of lubricin glycoforms in plasma/SF and their potential as biomarkers for disease. Five different lectins were used in the assay: Macrophage Galactose-type lectin (MGL),Sambucus NigraAgglutinin (SNA),Maackia AmurensisAgglutinin (MAA), Peanut Agglutinin (PNA), and Galectin-3 (Gal-3). Our results showed that the levels of lubricin glycoforms in late-stage knee OA plasma (typically <10 μg/ml) are 1-3 order of magnitude lower than in OA SF (typically> 100 μg/ml). Furthermore, plasma lubricin glycans displayed higher level of sialylation, while SF derived lubricin displayed higher level of Tn-antigens. In OA patients we found decreased level in plasma of SNA binding lubricin (p=0.0023) compared to controls. This lectin binds 6 linked sialic acid. In addition, we found that lubricin glycoforms correlated both with Body Mass Index (BMI) and age, especially in regards of sialylation as measured by both MAA and SNA. Our data suggest that glycosylation of lubricin is different comparing SF with plasma. Moreover, the glycosylation of plasma lubricin is altered in OA patients compared controls.