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Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated
C H King1, A Hull, P J Kleinhenz
1Department of Medicine, Case Western Reserve University, Cleveland, OH 44106.
Abstract:
Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.
Insights
This study identifies a specific serine proteinase in human neutrophils (PMN) that is activated during cell response. This enzyme, recognized by mAb 1-15, plays a role in PMN activation and has a molecular weight of 65,000-70,000.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Previous studies identified an activation-associated, chymotrypsin-like activity in human neutrophils (PMN) using anti-neutrophil/antichymotrypsin mAb 1-15.
- This activity was localized to the membrane fraction of isolated PMNs.
Purpose of the Study:
- To determine the molecular and biochemical characteristics of the mAb 1-15 antigen/proteinase.
- To elucidate the role of this proteinase in PMN activation.
Main Methods:
- Casein/acrylamide gel electrophoresis to assess proteinase activity.
- Reducing and nonreducing SDS-PAGE for molecular weight determination.
- Active site labeling with [3H]diisopropylfluorophosphate (DFP) to identify serine esterase activity.
- Substrate-affinity chromatography (phe-Sepharose, FMLP-Sepharose) for partial purification.
- Enzyme inhibition assays with various inhibitors.
- HPLC analysis and comparison with FMLP surface receptor.
Main Results:
- A Ca2(+)-dependent proteinase activity band (Mr 58,000-84,000) was detected in PMN membrane preparations.
- mAb 1-15 affinity purification recovered an enzymatically active, chymotrypsin-like antigen (Mr 65,000-70,000).
- Active site labeling confirmed a distinct membrane serine esterase (pI 6.3/Mr 65,000-70,000).
- The proteinase showed affinity for FMLP- or phenylalanine-binding proteins and was inhibited by specific serine proteinase inhibitors.
- Unlike the FMLP receptor, the DFP-labeled proteinase was not modified by endoglycosidase F.
Conclusions:
- The mAb 1-15 antigen is a distinct, active serine proteinase (Mr 65,000-70,000) present in human neutrophil membranes.
- This proteinase exhibits chymotrypsin-like activity and binds to substrates with aromatic amino acids.
- The identified proteinase appears to play a role in neutrophil (PMN) activation.