The red cell membrane contains calmodulin-regulated crosslinking and proteolytic activity

Area of Science:

• Biochemistry
• Cell Biology
• Hematology

Background:

• Transglutaminases (TGases) are enzymes involved in protein crosslinking.
• Erythrocyte TGase activity is known, primarily cytosolic forms active at high calcium.
• Membrane-associated TGases and their regulation remain less understood.

Purpose of the Study:

• To characterize a novel membrane-associated transglutaminase (TGase) activity in erythrocytes.
• To investigate the role of calmodulin (CaM) and calcium in regulating this erythrocyte TGase.
• To explore the potential function of this TGase in red blood cell membrane-cytoskeletal interactions.

Main Methods:

• Purification of membrane-associated TGase using CaM affinity chromatography.
• Assay of TGase activity at varying calcium concentrations (micromolar range).
• Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to identify protein bands.
• Inhibition studies using putrescine.

Main Results:

• Identified a membrane-associated TGase activity in erythrocytes requiring micromolar calcium and CaM.
• Purified TGase activity co-isolated with cytoskeletal components and showed a 10-fold increase post-purification.
• SDS-PAGE revealed two protein bands at 68,000 and 72,000 daltons.
• TGase crosslinked fibronectin, fibrinogen, and membrane cytoskeletal proteins, with associated degradation, inhibited by putrescine.

Conclusions:

• A novel CaM-calcium-regulated, membrane-associated erythrocyte TGase exists.
• This TGase may play a significant role in red blood cell membrane-cytoskeletal interactions.
• Further research is warranted to compare this TGase with those found in platelets and smooth muscle.