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

Abstract

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.

Day-to-day variation of urinary NGAL and rational for creatinine correction

“…Neutrophil gelatinase-associated lipocalin (NGAL) [1], also known as human neutrophil lipocalin (HNL) [2] and lipocalin-2, is a member of the lipocalin family of binding proteins and is in the circulation mainly produced by activated neutrophils. The main source of urinary NGAL (U-NGAL) is however tubular epithelial cells in the kidney [3].…”

The Origin of Multiple Molecular Forms in Urine of HNL/NGAL

“…The neutrophil granule release products were obtained as described (1,20). HK-2-conditioned supernatants at the 72-hour time point were harvested and supplemented with 0.1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, Germany) and Complete protease inhibitor cocktail tablets (Roche, Germany).…”

“…H uman neutrophil lipocalin(HNL) (1), also named neutrophil gelatinase-associated lipocalin (NGAL) (2), is a ubiquitous glycoprotein originally isolated from human neutrophils and localized in their specific granules. HNL/NGAL exists as a 25-kD monomer, or as a 45-kD disulfide-linked homodimer, and it is covalently conjugated with gelatinase (matrix metalloproteinase 9) via an intermolecular disulfide bridge as a 135-kD heterodimeric form (2).…”

Neutrophil Gelatinase–Associated Lipocalin in Dogs with Naturally Occurring Renal Diseases

“…Neutrophil gelatinase–associated lipocalin (NGAL), also called lipocalin 2, is a glycoprotein with a molecular weight of 25 kDa and a member of the lipocalin family 4. It was initially purified from neutrophils during infection and inflammation 5.…”