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Macrophage component gp160, a major trypsin-sensitive surface glycoprotein
Abstract:
Macrophages secrete a large number of proteases, implying in vivo exposure of the cell surface to proteolytic conditions. Mild trypsin treatment of 125I-labeled guinea pig peritoneal macrophages preferentially cleaves one surface component of apparent 160,000 mol wt. Similar trypsin treatment of macrophages with 3H-labeled carbohydrate surface moieties also cleaves a single 3H-labeled 160,000 mol wt glycoprotein, referred to as gp160. Nonreducing sodium dodecyl sulfate (SDS)-electrophoresis established that gp160 of trypsinized cells remains assembled in the membrane as a multichain disulfide-bonded molecule. gp160 was purified by detergent extraction, L. culinaris lectin affinity chromatography and DEAE-cellulose chromatography. The corresponding molecule from trypsinized cells was purified by the same procedure. Reducing SDS-electrophoresis of purified trypsinized 125I-labeled gp160 revealed two proteolytic fragments with apparent molecular weights of 85,000 and 71,000. Thus, mild trypsin treatment of macrophages preferentially cleaves a single surface protein, possibly at a single site. Because the two fragments of gp160 are accessible to lactoperoxidase and trypsin, both must be exposed on the membrane surface. The reactive carbohydrate site was found on the 85,000 mol wt fragment, which alone contains the 3H-label introduced into intact cells by neuraminidase, galactose, oxidase, and [3H]KBH4.
Insights
Macrophages have surface proteases, and mild trypsin treatment cleaves a 160,000 mol wt glycoprotein (gp160). This glycoprotein is exposed on the cell surface, with its carbohydrate moiety on an 85,000 mol wt fragment.
Area of Science:
- Cell Biology
- Proteomics
- Immunology
Background:
- Macrophages secrete proteases, indicating cell surface exposure to proteolytic conditions.
- Cell surface glycoproteins play crucial roles in cellular functions and interactions.
Purpose of the Study:
- To identify and characterize the primary surface protein cleaved by mild trypsin treatment on macrophages.
- To investigate the structure and surface accessibility of the cleaved glycoprotein.
Main Methods:
- Iodination (125I) and carbohydrate labeling (3H) of guinea pig peritoneal macrophages.
- Mild trypsin digestion and subsequent protein purification using lectin affinity and ion-exchange chromatography.
- Non-reducing and reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze protein fragments and molecular weights.
Main Results:
- Mild trypsin treatment preferentially cleaved a single surface glycoprotein (gp160) of 160,000 mol wt.
- gp160 remained assembled as a multichain disulfide-bonded molecule in the membrane after trypsinization.
- Reducing SDS-PAGE of purified gp160 yielded two fragments (85,000 and 71,000 mol wt), both accessible to the cell surface.
- The 85,000 mol wt fragment contained the carbohydrate label, indicating its surface exposure.
Conclusions:
- Macrophages possess a major surface glycoprotein (gp160) susceptible to mild trypsin cleavage.
- gp160 is exposed on the cell surface, with its carbohydrate moiety located on the larger fragment.
- This study provides insights into macrophage surface protein structure and proteolytic susceptibility.