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 pH 8.40, and the molecular weight 45 kDa (unreduced) or 24 kDa (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 coeffient was E1%,1cm = 13.76 at 280 nm. The 60 amino acid sequence revealed up to 65% sequence homology with rat alpha 2-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 microgram 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.
Xu
S Y
SY
Department of Clinical Chemistry, University Hospital, Uppsala, Sweden.
Carlson
M
M
Engström
A
A
Garcia
R
R
Peterson
C G
CG
Venge
P
P
D000209:Acute-Phase Proteins; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007120:Immunochemistry; D007700:Kinetics; D000071068:Lipocalin-2; D054834:Lipocalins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000494:pharmacology; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry* / Q000648:ultrastructure; D015513:Oncogene Proteins; D011480:Protease Inhibitors; D011518:Proto-Oncogene Proteins; D012016:Reference Values; D013347:Subcellular Fractions / Q000737:chemistry
D000209:Acute-Phase Proteins; D000596:Amino Acids; D002352:Carrier Proteins; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D015513:Oncogene Proteins; D011480:Protease Inhibitors; D011518:Proto-Oncogene Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase