Purification and characterization of a human neutrophil lipocalin (HNL) from the secondary granules of human neutrophils

• A 45 kDa-protein was purified from the granules of human neutrophils. The protein consists of two apparently identical subunits. The isoelectric point was pH8.40, and the molecular weight 45kDa (unreduced) or 24kDa (reduced). Treatment of the protein with Endoglucosidase F resulted in a reduction in the molecular weight to 20 kDa, indicating the presence of N-linked carbohydrate. The extinction coefficient was E1%lcm = 13.76 at 280nm. The 60 amino acid sequence revealed up to 65% sequence homology with rat α2-microglobulin-related protein, which belongs to the lipocalin family. The protein co-sedimented with secondary (specific) granule marker proteins and correlated to the neutrophil content of Lactoferrin (r = 0.81,p<0.001) and was estimated to be 0.59 fig 10-6 cells. Release studies showed that the neutrophils released 51.4 ± 9.0% of the total cellular content of the protein when they were exposed to serum-opsonized particles, which was much higher than the release of Myeloperoxidase (12.7 ±3.5%) and Lactoferrin (22.9 ± 4.7%). The N-terminal and four tryptic fragment amino acid sequence of the protein was identical with an N-formyl peptide binding 24 kDa protein and gelatinase associated protein of human neutrophils. In conclusion, we have purified and characterized a protein, human neutrophil lipocalin (HNL), from the secondary granules of human neutrophils and shown that it is readily mobilized from the neutrophils upon stimulation.

Nyckelord

Acute-Phase Proteins

Amino Acid Sequence

Amino Acids/analysis

Carrier Proteins/*isolation & purification/secretion

Cytoplasmic Granules/*chemistry/secretion

Humans

Hydrolysis

Immunochemistry

Kinetics

Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase/pharmacology

Molecular Sequence Data

Neutrophils/*chemistry/ultrastructure

Oncogene Proteins

Protease Inhibitors

Reference Values

Research Support; Non-U.S. Gov't

Subcellular Fractions/chemistry

MEDICINE

MEDICIN

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