**2.2.1 Enrichment of synovial lubricin**

Synovial lubricin is a heavily negatively charged glycoprotein that can be enriched by ionexchange chromatography (Fig. 1A). Lubricin containing fractions which were determined by sandwich ELISA were pooled and precipitated by 80% ethanol. The amount of protein in these fractions corresponded to 1.38 mg/mL synovial fluid (mean, n=5). Considering that lubricin has been shown to be in the range of 0.2-0.5 mg/mL(Marcelino et al., 1999; Schmid et al., 2001; Elsaid et al., 2005), this indicates that additional proteins (*e.g.*, albumin) and glycoproteins (*e.g.*, fibronectin, aggrecan) are co-purified (See proteomic section).

Fig. 1. Enrichment of human lubricin from synovial fluid. (A) Representative elution profile of DEAE ion-exchange chromatography. Protein levels in each fraction were determined by BCA method, while lubricin-containing fractions (Fr. 35-41) were pooled and precipitated with 80% ethanol. (B) Reduced and alkylated synovial fluid (SF, 1 µL) and enriched sample (Lubricin, 2 µg) were separated by Ag-PAGE. Protein bands were visualized by silver nitrate or detected by Western blot (WB).

As shown in Fig. 1B, silver staining of Ag-PAGE showed one major band around 300 kDa indicating that the majority of synovial proteins were removed during enrichment. When more samples were loaded, additional faint bands were also detected (Fig. 4B-2). Both mouse monoclonal (Fig. 1B) and rabbit polyclonal antibody (not shown) specifically react to prepared lubricin, respectively. Though there are few bands smaller than lubricin, none of them reacted with lubricin-specific antibodies.

#### **2.2.2 Glyco-epitope on synovial lubricin verified by immunoassay**

To examine the glycan profile on lubricin, purified samples were primarily analyzed by immunoassay with lectins or anti-carbohydrate antibodies (Fig. 2). Synovial lubricin was positive to the lectins specific to sialic acid and T antigen, such as WGA (sialic acid and terminal GlcNAcβ1,4), MAA-I (specific to α2,3-linked sialic acid), and PNA (T antigen, Galβ1,3GalNAcα1-*O*-Ser/Thr). Lubricin also reacted with HAA, a lectin specific to terminal GalNAcα1- including Tn antigen (GalNAcα1-*O*-Ser/Thr). Lectin immunoblot of synovial lubricin was also negative to ConA, which binds to branched Manα1- on high-mannose and

Synovial lubricin is a heavily negatively charged glycoprotein that can be enriched by ionexchange chromatography (Fig. 1A). Lubricin containing fractions which were determined by sandwich ELISA were pooled and precipitated by 80% ethanol. The amount of protein in these fractions corresponded to 1.38 mg/mL synovial fluid (mean, n=5). Considering that lubricin has been shown to be in the range of 0.2-0.5 mg/mL(Marcelino et al., 1999; Schmid et al., 2001; Elsaid et al., 2005), this indicates that additional proteins (*e.g.*, albumin) and

Fig. 1. Enrichment of human lubricin from synovial fluid. (A) Representative elution profile of DEAE ion-exchange chromatography. Protein levels in each fraction were determined by BCA method, while lubricin-containing fractions (Fr. 35-41) were pooled and precipitated with 80% ethanol. (B) Reduced and alkylated synovial fluid (SF, 1 µL) and enriched sample (Lubricin, 2 µg) were separated by Ag-PAGE. Protein bands were visualized by silver nitrate

As shown in Fig. 1B, silver staining of Ag-PAGE showed one major band around 300 kDa indicating that the majority of synovial proteins were removed during enrichment. When more samples were loaded, additional faint bands were also detected (Fig. 4B-2). Both mouse monoclonal (Fig. 1B) and rabbit polyclonal antibody (not shown) specifically react to prepared lubricin, respectively. Though there are few bands smaller than lubricin, none of

To examine the glycan profile on lubricin, purified samples were primarily analyzed by immunoassay with lectins or anti-carbohydrate antibodies (Fig. 2). Synovial lubricin was positive to the lectins specific to sialic acid and T antigen, such as WGA (sialic acid and terminal GlcNAcβ1,4), MAA-I (specific to α2,3-linked sialic acid), and PNA (T antigen, Galβ1,3GalNAcα1-*O*-Ser/Thr). Lubricin also reacted with HAA, a lectin specific to terminal GalNAcα1- including Tn antigen (GalNAcα1-*O*-Ser/Thr). Lectin immunoblot of synovial lubricin was also negative to ConA, which binds to branched Manα1- on high-mannose and

glycoproteins (*e.g.*, fibronectin, aggrecan) are co-purified (See proteomic section).

**2.2 Result** 

**2.2.1 Enrichment of synovial lubricin** 

or detected by Western blot (WB).

them reacted with lubricin-specific antibodies.

**2.2.2 Glyco-epitope on synovial lubricin verified by immunoassay** 

hybrid type *N*-glycans, suggesting *N*-glycans were absent on lubricin or in very low amounts. The same negative results were obtained with *Aleuria aurantia* lectin (AAL), which recognized both peripheral and core fucosylated glycans.

Fig. 2. Glyco-epitope on lubricin analyzed by immunoblot. Reduced and alkylated enriched lubricin sample (6 µg/lane) was separated by 3-8% Tris/acetate NuPAGE and blotted to PVDF membrane. Strips were incubated with various lectins or anti-carbohydrate antibodies after blocking with 1% BSA in TBS-T buffer. After incubating with HRP conjugated corresponding secondary antibodies and streptavidin, bands were developed by SuperSignal West Femto maximum sensitivity substrate. CB, Coomassie blue stained gel; PNA, peanut agglutinin; WGA, wheat germ agglutinin; AAL, *Aleuria aurantia* lectin; HAA, *Helix aspersa* agglutinin; Anti-sLex, sialyl Lewis x-specific antibody; T, T antigen, Galβ1,3GalNAc-*O*-Ser/Thr; Tn, Tn antigen, GalNAc-*O*-Ser/Thr.

When synovial lubricin was investigated by anti-carbohydrate antibodies (Fig. 2), lubricin was suggested to have T antigen and sialyl Lewis x (sLex, structure in Fig. 3C). Western blot showed (data not presented) that lubricin was negative for anti-carbohydrate antibodies specific to chondroitin sulfate (mAb CS56), sLea [NeuAcα2,3Galβ1,3(Fucα1,4)GlcNAcβ1-], (mAb CA19-9), 3'-sulfo-Lea [NeuAcα2,3Gal(3S)β1,3(Fucα1,4)GlcNAc-], Leb [Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ1- ], (mAb 2-25LE], MECA-79 epitopes, Tn antigen (mAb 5F4 and 1E3), and sialyl Tn antigen [NeuAcα2,6GalNAc-*O*-Ser/Thr], (mAb TKH2 and 3F1). Results obtained from anticarbohydrate antibodies agree with results from the lectin immunoblot except for lectin HAA. Together with the lectin immunoblot, these results demonstrated synovial lubricin had sialylated glycans, core 1 *O*-glycan and peripheral sLex epitope. In order to reveal the identity of the sLex containing *O*-glycans and identify other glycan epitopes not recognized by the antibodies and lectin used, additional experiments were carried out.
