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C. Horst, J.C. Besharse; Characterization of a 425 kD Sialated Glycoconjugate Associated with the Y–shaped Cross–Linkers of the Connecting Cilium. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3642.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Bridging the photoreceptor inner and outer segments, the connecting cilium functions as both a conduit for, and blockade of, transport. How these functions are accomplished remains unknown. Due to their location, Y–shaped crosslinkers that attach connecting cilium axonemal microtubules to the overlying plasma membrane are thought to participate in one or both of these processes. Through binding of a specific monoclonal antibody, K26, and lectin characterization, previous work identified a 425 kD glycoconjugate as a detergent–stable component of the crosslinkers bearing sialic acid–Gal–GalNAc–Ser/Thr mucin–like O–linked oligosaccharies. Further characterization of the 425 kD glycoconjugate may suggest its function. Methods: The detergent–extracted photoreceptor cytoskeleton subcellular fraction, highly enriched in axonemes, was treated with neuraminidase followed by: 1) immunofluorscence or Western blot analysis to detect the K26 antigen or lectin binding sites, and 2) electron microscopy to visualize the structure of the connecting cilium. Results: Removal of sialic acid by neuraminidase reveals PNA binding to axonemes and does not remove K26 binding. Once revealed, PNA binding blocks the binding of K26. PNA does not block binding of an antibody to the axonemal protein MRJ, nor does the lectin GNA block the binding of K26. Thus, the interference is specific to PNA and K26. Attempts to double–label axonemes with PNA and K26 showed only PNA binding regardless of the application order suggesting PNA has a higher affinity than K26. Sialic acid plays a large role in the structure of the 425 kD glycoconjugate. Neuraminidase prior to Western blot analysis results in a shift of the 425 kD glycoconjugate to 62 kD as determined by both K26 and PNA binding. This large shift is characteristic of mucins where sugars can comprise up to 90% of the molecular mass. Electron microscopy shows that a halo devoid of heavy–metal staining surrounding the axonemes in the region of the crosslinkers, is diminished after neuraminidase treatment. Conclusions: K26 and PNA binding sites on the 425 kD glycoconjugate are overlapping or adjacent. The 425 kD glycoconjugate bears a large amount of sialic acid that contributes to an extracellular collar surrounding the connecting cilium. The collar may act to isolate the plasma membrane of the connecting cilium, protecting components in transit to the outer segment. Alternatively, the collar may act to maintain the connecting cilium plasma membrane integrity, preventing fusion of the inner and outer segments.
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