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Cell-free synthesis and processing of the heavy and light chains of HLA-DR antigens
Abstract:
Antisera have been prepared against the separated glycoprotein chains (p29 and p34) of HLA-DR antigens (p29,34) isolated from membranes of the B lymphoblastoid cell line JY (DRw4,6). These antisera (anti-p29 and anti-p34) were characterized by immunoprecipitation of in vivo labeled, detergent-solubilized extracts of JY cells grown in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation. Anti-p29 and anti-p34 specifically immunoprecipitated the precursors to p29 and p34, respectively, from the cell-free translation products of a rabbit reticulocyte lysate system supplemented with polyadenylic acid-containing messenger RNA (mRNA) isolated from JY cells. These precursors, pre-p34 and two pre-p29s, appeared to be 1,500-3,000 daltons larger than their counterparts from tunicamycin-treated cells and presumably were synthesized with N-terminal extensions (signal sequences). Processing of pre-p29 and pre-p34 occurred during cell-free translation in the presence of dog pancreatic microsomes, which resulted in cleavage of the polypeptide, addition of glycan moieties, and segregation of the two chains in the cisternal portion of the microsome. the precursors of p29 and p34 were not appreciably different in isoelectric points on two-dimensional gels from p26 and p28, the light and heavy chains from antigens arise from separate mRNA, and not as a single polypeptide, which is endoproteolytically cleaved.
Insights
Researchers developed antisera against HLA-DR glycoprotein chains (p29, p34). These antibodies identified precursor proteins, revealing that HLA-DR heavy and light chains originate from separate messenger RNAs, not a single precursor.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Human Leukocyte Antigen (HLA) class II molecules, specifically HLA-DR, are crucial for immune responses.
- Understanding the synthesis and processing of HLA-DR subunits is vital for comprehending immune regulation and disease.
Purpose of the Study:
- To generate specific antisera against the individual glycoprotein chains of HLA-DR antigens.
- To investigate the synthesis and processing pathways of HLA-DR p29 and p34 chains.
- To determine if HLA-DR chains originate from separate messenger RNAs or a single precursor.
Main Methods:
- Preparation and characterization of antisera against p29 and p34 glycoprotein chains.
- Immunoprecipitation of in vivo labeled cell extracts and cell-free translation products.
- Analysis of protein precursors using tunicamycin to inhibit glycosylation.
- Two-dimensional gel electrophoresis to compare precursor and mature protein isoelectric points.
Main Results:
- Specific antisera (anti-p29, anti-p34) were successfully generated and characterized.
- Precursor forms of p29 and p34 (pre-p29, pre-p34) were identified, larger than mature chains due to signal sequences.
- Cell-free translation with microsomes demonstrated processing, including signal peptide cleavage and glycosylation.
- Evidence suggests that the precursors of p29 and p34 originate from separate messenger RNAs.
Conclusions:
- The study confirms that HLA-DR heavy and light chains are synthesized from distinct messenger RNAs.
- The findings elucidate the early steps of HLA-DR biosynthesis, including signal peptide processing and translocation.
- This research provides a foundation for further studies on HLA-DR assembly and its role in immune function.