Anti-lubricin monoclonal antibodies created using lubricin-knockout mice immunodetect lubricin in several species and in patients with healthy and diseased joints

Abstract

Lubricin, encoded by the gene PRG4, is the principal lubricant in articulating joints. We immunized mice genetically deficient for lubricin (Prg4-/-) with purified human lubricin, and generated several mAbs. We determined each mAb's binding epitope, sensitivity, and specificity using biologic samples and recombinant lubricin sub-domains, and we also developed a competition ELISA assay to measure lubricin in synovial fluid and blood. We found the mAbs all recognized epitopes containing O-linked oligosaccharides conjugated to the peptide motif KEPAPTTT. By western blot, the mAbs detected lubricin in 1 μl of synovial fluid from several animal species, including human. The mAbs were specific for lubricin since they did not cross-react with other synovial fluid constituents from patients with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), who genetically lack this protein. The competition ELISA detected lubricin in blood samples from healthy individuals but not from patients with CACP, indicating blood can be used in a diagnostic test for patients suspected of having CACP. Lubricin epitopes in blood do not represent degradation fragments from synovial fluid. Therefore, although blood lubricin levels did not differentiate patients with inflammatory joint disease from healthy controls, epitope-specific anti-lubricin mAbs could be useful for monitoring disease activity in synovial fluid.

Fig 1. Western blots probed with anti-lubricin mouse monoclonal antibodies (mAbs) 9g3, 7h12, 5c11, 6a8, and 8e3 detect lubricin in synovial fluid but do not detect other mucins.

(A) Five μg of human lubricin purified from synovial fluid (PHL), and 1 μl of synovial fluid recovered from patients with osteoarthritis (OA), CACP, or from bovine, porcine, goat, dog, rat and guinea pig were electrophoresed under reducing conditions on 4–20% SDS-PAGE gradient gels, transferred to PVDF, and immunodetected using the biotin labeled mAbs. Note that the mAbs detect protein in the synovial fluid from patients with OA and from other species, but not from the patient with CACP, who is genetically deficient for lubricin. (B) 0.7 μg of Purified human lubricin (PHL), porcine gastric mucin (PM) and bovine submaxillary mucin (BSM) were electrophoresed under non-reducing conditions on 4–12% SDS-PAGE gradient gels, transferred to nitrocellulose and immunodetected using the mAbs. For mAb-8e3, 1.4 μg of PHL was loaded. Note that anti-lubricin mAbs did not cross react with BSM or PM.

Fig 2. The mAbs detect an octapeptide motif present in the first mucin-like domain of human lubricin.

Different domains of lubricin (depicted in the top panel beneath a schematic of the protein and its 12 coding exons) were cloned into a mammalian expression construct downstream of a signal peptide sequence and a Hemaglutinin epitope-tag (HA) sequence. Domain Mu1b was also fused to a human IgG-Fc fragment (Mu1b-Fc). An octapeptide motif (KEPAPTTT), which occurs multiple times within the first mucin-like domain (Mu1), was also fused to the IgG-Fc fragment (T-Fc). These constructs were transiently transfected into 293T cells. Serum-free conditioned media were collected and subjected to SDS-PAGE on 4–20% gradient gels. Anti-HA antibody detected all recombinant proteins in the media (HA), although weak immunodetectable bands occurred for Mu1a and Mu1c. Monoclonal antibodies 9g3, 7h12, 5c11 and 6a8 detected the secreted first mucin domain Mu1, Mu1a and Mu1c, but not Mu1b or the second mucin domain Mu2. Mu1b is the first 98 AA of the first mucin domain and does not contain KEPAPTTT; instead it has “KEPTPTT” and other “ETTT”-containing repeats. The only peptide motifs that Mu1a and Mu1c share is: “KEPAPTTP”. Antibodies 9g3, 7h12 and 6a8 also strongly recognized the KEPAPTTT containing recombinant protein (T-Fc), while 5c11 weakly interacted with T-Fc. Horseradish peroxidase (HRP) conjugated anti-mouse IgG, which was used as secondary antibody to detect 9g3, showed weak cross reactivity to human IgG-Fc. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled 7h12, 5c11 and 6a8, and showed no cross-reactivity by itself (data not shown).

Fig 3. The mAbs recognize an epitope that contains an O-linked glycan modification of the octapeptide K-E/A-P-A-P-T-T-T/A/P.

Serum-free conditioned media from 293T cells expressing T-Fc (i.e., KEPAPTTT) recombinant protein or variants of this octapeptide motif were resolved on reducing SDS-PAGE gel and immunodetected with the mAbs. (A) Recombinant T-Fc immunodetected using antibodies indicated in each panel. Removal of O-linked glycans by enzymatic digestion with neuraminidase and O-glycosidase caused loss or attenuated recognition by all mAbs, except anti-HA antibody. Interestingly, all mAbs showed significantly increased immunoreactivity to the recombinant protein treated with neuraminidase alone, suggesting sialic acid modifications that usually occur at sugar chain termini interfere with epitope-antibody interactions. (B) Recombinant HA-KEPAPTTT-Fc (T-Fc) and HA-IgG-Fc (Fc) proteins were treated by neuraminidase and/or O-glycosidase and immunodetected using anti-human IgG-Fc. Note sugar modification on the KEPAPTTT peptide, but not on the Fc fragment, is responsible for changes in polypeptide mobility upon deglycosylation. (C) Recombinant octapeptides fused downstream of HA and upstream of IgG-Fc immunodetected using antibodies indicated in each panel. Anti-HA antibody showed that all mutant recombinant proteins were expressed. Recombinant protein HA-KEPAPTTT-Fc (1) was immunodetected by all monoclonal antibodies. These antibodies did not detect the variant forms of the recombinant protein: HA-KEPAPTAT-Fc (2); HA-KEPAATTT-Fc (3); HA-KEPAPTT-Fc (4); HA-PAPTTT-FC (5). Note that the anti-mouse IgG, used as secondary antibody for mAb-9g3, showed strong cross-reactivity with the human IgG-Fc present in all recombinant proteins. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled mAbs 7h12, 5c11 and 6a8, and showed no background by itself (data not shown). (D) Schematic depicting the peptide and O-linked glycosylation motifs that occur commonly in the mucin-like domain of human lubricin. Sialylated and non-sialylated O-linked oligosaccharides can be added to the Threonine (T) residues; two potential oligosaccharides (sialylated and non-sialylated) are indicated and their relative affinities to the mAbs are indicated by the arrow weights.

Fig 4. mAb-7h12 can measure lubricin in human plasma, serum, and synovial fluid by competition ELISA.

(A) Photograph showing the result of a competition ELISA performed in triplicate. Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with purified lubricin (the concentrations of purified lubricin are indicated on top of each column) and added to lubricin-coated wells. After several washes, the mAb bound to the lubricin-coated wells, was detected colorimetrically using horseradish peroxidase (HRP) conjugated to streptavidin. Note when antibody is not pre-incubated with lubricin (0 μg/ml), all antibody binds to the pre-coated wells and the HRP-streptavidin detection of bound antibody turns these wells dark blue. In contrast, pre-incubating the antibody with increasing amounts of purified lubricin reduces antibody binding to the wells and there is less color formation by HRP-streptavidin. (B) Standard curve derived from the O.D. 415 nm values for the competition ELISA shown in panel A. The x-axis indicates the concentration of lubricin (μg/ml) that was pre-incubated with the antibody; the y-axis indicates the average difference in O.D. reading compared to the 0 μg/ml lubricin control. (C) Photograph showing the result of a competition ELISA performed in duplicate (individual rows). Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with 1:50 dilutions of human plasma or serum, or 1:50,000 dilutions of human synovial fluid. The plasma samples are from patients with CACP (CA697–1, CA698–1, CA698–2) and their unaffected family members (CA697–2, CA698–4, CA698–5). The serum samples are from healthy controls (OT701–1; OT702–1). The synovial fluid samples are from patients with OA, RA, or CACP (CA). Note that plasma and synovial fluid from patients with CACP do not reduce antibody binding to lubricin-coated wells as indicated by the dark blue color, whereas plasma, serum, or synovial fluid from unaffected individuals does reduce antibody binding to the lubricin-coated wells. Based upon the measured O.D. values for these samples (data not shown), no detectable lubricin is present in serum and synovial fluid from patients with CACP, whereas ~ 200 μg/ml is present in the RA and OA synovial fluid samples and ~ 0.2 μg/ml is present in the plasma and serum samples from unaffected relatives and controls. (D) mAbs 9g3, 5cll, 6a8 and 7h12 can be used interchangeably with recombinant human lubricin in a competition ELISA format.

Fig 5. Immunoprecipitation of lubricin from human serum, plasma and synovial fluid.

Biotin-labeled mAb-7h12 was mixed with plasma samples from a patient with CACP (CA697–1) or his unaffected father (CA697–2), serum samples from a patient with rheumatoid arthritis (RA) or an age- and sex-matched healthy control (Control), synovial fluid samples from a patient with osteoarthritis (OA), rheumatoid arthritis (RA), or CACP (CA), and with human (hLub-IP) and bovine (bLub-IP) lubricin that had been purified from synovial fluid. Antibody was recovered using streptavidin beads and proteins co-precipitated with mAb-7h12 were eluted, subjected to SDS-PAGE, transferred to PVDF, and immunodetected with mAb-7h12 (upper panel) or polyclonal Ab-J108N (lower panel). Note mAb-7h12 immunoprecipitated lubricin from plasma and serum that is also recognized by polyclonal antibody J108N. In contrast, lubricin immunoprecipitated from OA and RA synovial fluid is not recognized by polyclonal antibody J108N. J108N also does not recognize immunoprecipitated purified human lubricin (hLub-IP). The is no non-specific binding of lubricin to streptavidin beads as demonstrated by the lack of immunodetectable protein when mAb-7h12 is not used in the co-IP (no Ab control) of plasma from the unaffected CACP parent or synovial fluid from a patient with RA. As positive controls for immunodetection purified human and bovine lubricin (hLub and bLub) were directly subjected to SDS-PAGE for immunodetection.

Fig 6. Immunodetection of lubricin in synovial tissue.

Photomicrographs of tissue sections from archival human synovial biopsies from patients with osteoarthritis (A) or CACP (B). Sections had been incubated with mAb-7h12 and detected with avidin/biotin/peroxidase. Note immunodetectable lubricin (brown-colored stain) is concentrated along the surface of OA synovium, and not found in CACP synovium. The OA sample was counterstained with hematoxylin & eosin and imaged at 1000X magnification. The CACP sample was counterstained with eosin and imaged at 600X magnification.