Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multistep pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 congenital disorders of glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multispanning membrane protein located in the ER, with its N and C termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

Near-infrared light-boosted antimicrobial activity of minocycline/hyaluronan/carbon nanohorn composite toward peri-implantitis treatments.

Dental implant therapy is a reliable treatment for replacing missing teeth. However, as dental implants become more widely used, peri-implantitis increasingly has become a severe complication, making successful treatment more difficult. As a result, the development of effective drug delivery systems (DDSs) and treatments for peri-implantitis are urgently needed. Carbon nanohorns (CNHs) are carbon nanomaterials that have shown promise for use in DDSs and have photothermal effects. The present study exploited the unique properties of CNHs to develop a phototherapy employing a near-infrared (NIR) photoresponsive composite of minocycline, hyaluronan, and CNH (MC/HA/CNH) for peri-implantitis treatments. MC/HA/CNH demonstrated antibacterial effects that were potentiated by NIR-light irradiation, a property that was mediated by photothermal-mediated drug release from HA/CNH. These antibacterial effects persisted even following 48 h of dialysis, a promising indication for the clinical use of this material. We propose that the treatment of peri-implantitis using NIR and MC/HA/CNH, in combination with surgical procedures, might be employed to target relatively deep affected areas in a timely and efficacious manner. We envision that this innovative approach will pave the way for future developments in implant therapy.

Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.