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Delayed activation of the mannose receptor following synthesis. Requirement for exit from the endoplasmic reticulum
S E Pontow1, J S Blum, P D Stahl
1Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. pstahl@cellbio.wustl.edu
Abstract:
The macrophage mannose receptor specifically recognizes proteins and particles bearing mannose terminal oligosaccharide chains. In the present study, we examined the ability of newly synthesized receptor to bind ligand. Human monocyte-derived macrophages were pulse-labeled with [35S]Met and prepared for affinity chromatography on mannose-Sepharose. Mannose receptor in the flow-through and eluted fractions was detected by fluorography following immunoprecipitation and gel electrophoresis. Labeled mannose receptor was found exclusively in the nonbinding fraction until 10 min of chase. Following a 60-min chase, 67-86% of newly synthesized receptor was precipitated from the bound column fraction. The half-time for development of receptor binding activity was determined to be 35-40 min compared with a 45-min half-time for development of endoglycosidase H resistance. Mannose receptor synthesized by cells incubated in brefeldin A required more than 120 min to acquire endoglycosidase H resistance and maximal binding activity. Inhibitors of N-linked oligosaccharide processing or of O-glycosylation had no effect on the development of mannose receptor binding activity. Monensin prevented terminal sialylation of oligosaccharide side chains but did not inhibit receptor activation. Inclusion of aluminum fluoride in the chase media reversibly inhibited development of endoglycosidase H resistance and mannose-binding activity. We conclude that the mannose receptor undergoes delayed activation following synthesis and suggest that the activating event(s) occur following exit of the receptor from the endoplasmic reticulum and prior to its entry into the trans-Golgi.
Insights
The macrophage mannose receptor requires time after synthesis to become active in binding ligands. This delayed activation process is linked to its post-translational modifications and transport through cellular compartments.
Area of Science:
- Immunology
- Cell Biology
- Glycobiology
Background:
- The macrophage mannose receptor (MMR) is crucial for innate immunity, recognizing mannose-terminated structures on pathogens and host molecules.
- Understanding the synthesis and activation of MMR is vital for comprehending immune responses and developing targeted therapies.
Purpose of the Study:
- To investigate the temporal aspects of newly synthesized mannose receptor activation and ligand-binding capability.
- To elucidate the post-translational modifications and cellular trafficking events involved in MMR functional maturation.
Main Methods:
- Human monocyte-derived macrophages were pulse-labeled with [35S]Met and subjected to affinity chromatography using mannose-Sepharose.
- Receptor binding activity was assessed by fluorography after immunoprecipitation and gel electrophoresis.
- Endoglycosidase H resistance and effects of various inhibitors (brefeldin A, monensin, aluminum fluoride) were evaluated.
Main Results:
- Newly synthesized mannose receptor was initially unable to bind ligand, with binding activity developing over 35-40 minutes.
- This activation occurred concurrently with the acquisition of endoglycosidase H resistance (45-minute half-time).
- Inhibitors of N-linked and O-linked glycosylation did not affect activation, but brefeldin A and aluminum fluoride significantly delayed it.
Conclusions:
- The mannose receptor undergoes a significant delay between synthesis and the acquisition of ligand-binding activity.
- Receptor activation is linked to post-endoplasmic reticulum and pre-trans-Golgi processing events.
- The findings suggest a regulated maturation process for the mannose receptor before its cell surface expression and function.