Proteoglycan 4 (lubricin) is a highly sialylated glycoprotein associated with cardiac valve damage in animal models of infective endocarditis

Abstract

Streptococcus gordonii and Streptococcus sanguinis are primary colonizers of tooth surfaces and are generally associated with oral health, but can also cause infective endocarditis (IE). These species express "Siglec-like" adhesins that bind sialylated glycans on host glycoproteins, which can aid the formation of infected platelet-fibrin thrombi (vegetations) on cardiac valve surfaces. We previously determined that the ability of S. gordonii to bind sialyl T-antigen (sTa) increased pathogenicity, relative to recognition of sialylated core 2 O-glycan structures, in an animal model of IE. However, it is unclear when and where the sTa structure is displayed, and which sTa-modified host factors promote valve colonization. In this study, we identified sialylated glycoproteins in the aortic valve vegetations and plasma of rat and rabbit models of this disease. Glycoproteins that display sTa vs. core 2 O-glycan structures were identified by using recombinant forms of the streptococcal Siglec-like adhesins for lectin blotting and affinity capture, and the O-linked glycans were profiled by mass spectrometry. Proteoglycan 4 (PRG4), also known as lubricin, was a major carrier of sTa in the infected vegetations. Moreover, plasma PRG4 levels were significantly higher in animals with damaged or infected valves, as compared with healthy animals. The combined results demonstrate that, in addition to platelet GPIbα, PRG4 is a highly sialylated mucin-like glycoprotein found in aortic valve vegetations and may contribute to the persistence of oral streptococci in this protected endovascular niche. Moreover, plasma PRG4 could serve as a biomarker for endocardial injury and infection.

Keywords: O-linked glycan; ITIH4; cardiovascular; sialic acid; thrombus.

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Figures

Fig. 1

Recombinant streptococcal lectins and sialoglycan structures. The SLBRs are derived from Streptococcus gordonii serine-rich repeat adhesins and recognize different subsets of α2–3 sialylated O-glycans (Bensing et al. 2018). SLBR-B is from GspB; SLBR-H is from Hsa; SLBR-N is from the UB10712 homolog (formerly referred to as the Streptococcus mitis NCTC10712 homolog). The high-affinity synthetic ligands were identified by array and ELISA studies (Bensing et al. 2016, 2019b). The O-glycan subsets were inferred from affinity capture and O-glycan profiling experiments and from cell-based mucin sialoglycan display libraries (Bensing et al. 2018; Narimatsu et al. 2019). The core 2 glycan extensions may include poly-lactosamine, fucose and sulfate.

Fig. 2

Lectin and western blotting of Streptococcus gordonii-infected rat aortic valve vegetations. All animals were catheterized 3 days prior to infection with S. gordonii, and the transaortic (TA) catheters remained in place for the duration of the experiment. Vegetations were harvested from three animals 1 h after infection (on day 3) or three animals 72 h after infection (on day 6) as indicated. Lanes contain 5 μg homogenized vegetation proteins. Parallel samples were either stained with Coomassie or were transferred to nitrocellulose and probed with antibodies or SLBRs as indicated.

Fig. 3

Lectin and western blotting of plasma from IE rats. Plasma from the set of animals from which vegetations were collected and analyzed in Figure 2. Lane 7 (P) contains plasma prepared from commercial Sprague–Dawley rat blood. Lanes contain 0.4 μL of plasma. Samples were probed with antibodies or SLBRs as indicated. Note that the

Fig. 4

Longitudinal analysis of rat plasma pre- and postinfection. (A) Quantitative analysis of plasma PRG4 from eight animals, collected at three time-points during the experiment: (a) prior to catheterization; (b) 3 days postcatheterization, prior to infection; (c) 1 h after infection with Streptococcus gordonii. Bar indicates mean column value. Statistical significances were determined by two-way ANOVA, followed by Tukey’s post hoc for multiple comparisons. Asterisk indicates P < 0.05. (B) Lectin and western blotting of plasma from three representative animals. Lanes contain 0.4 μL plasma, and blots were probed with anti-PRG4 or SLBR-H. Note that the <100 kDa proteins seen in the PRG4 blot were detected with the secondary anti-mouse IgG/IgM alone and are therefore not PRG4 fragments.

Fig. 5

Lectin and western blotting of healthy, NBTE and IE rat aortic valve tissue. Samples were collected from healthy animals (1–3), animals catheterized 6 days (4 and 5) or animals catheterized 6 days and infected with Streptococcus gordonii on day 3 (6–8). Lanes contain 5 μg homogenized tissue. Parallel samples were either stained with Coomassie or transferred to nitrocellulose and probed with anti-PRG4 or SLBR-H as indicated.

Fig. 6

O-glycans on PRG4 from Streptococcus gordonii-infected rat aortic valve vegetations. Upper and lower panels have O-glycans released from PRG4 recovered from the vegetations of two different animals (from 500 μg homogenized vegetation proteins). Asterisks (*) indicate polyhexose O-glycans that were omitted from the pie charts and O-glycan profile tables.

Fig. 7

Comparison of PRG4 isolated from intact vs. infected rat valves. PRG4 was enriched from aortic valve tissue pooled from three healthy animals (upper) or three IE animals (lower). The O-glycans were released from the total PRG4 captured from ~1 mg valve tissue. Asterisks (*) indicate polyhexose O-glycans that were omitted from the pie charts and O-glycan profile tables.

Fig. 8

Profile of O-glycans released from rat plasma PRG4. Plasma was collected from the same animal precatheterization (upper panel), 3 days postcatheterization (middle panel) and 72 h postinfection with Streptococcus gordonii (lower panel). The profiles correspond to the total PRG4 recovered from 60 μL plasma.

Fig. 9

Sialylated glycoproteins in rabbit plasma and aortic valve tissue. (A) Valve tissue was pooled from three untreated (7–9), NBTE (4–6) or IE (1–3) animals. Proteins were either stained with Coomassie or were transferred to nitrocellulose and probed with anti-PRG4, SLBR-H or SLBR-N as indicated. Lanes contain 5 μg homogenized proteins. (B) Longitudinal analysis of plasma collected prior to catheterization (day 0), 3 days postcatheterization and 6 days postcatheterization and uninfected (NBTE) or infected with Streptococcus gordonii on day 3 (IE). Lanes contain 0.4 μL plasma. Blots were probed with anti-PRG4 or a combination of SLBR-H and -N for the detection of sialylated core 1 and core 2 glycans.