Characterization of bovine neutrophil gelatinase-associated lipocalin.
A protein of relative molecular mass of approximately 25,000 was purified from bovine colostrum by cation-exchange and size-exclusion chromatography. The N-terminus of the protein matched the sequence predicted by the National Center for Biotechnology Information for the bovine homolog of human neutrophil gelatinase-associated lipocalin, a glycoprotein of relative molecular mass 25,000 belonging to the family of lipocalins. The protein was further designated as bovine neutrophil gelatinase-associated lipocalin (bNGAL). Sodium dodecyl sulfate-PAGE of enzymically deglycosylated bNGAL indicated that the intact protein bears one N-linked glycan. Monosaccharide and mass spectrometric analyses of released N-linked carbohydrates revealed the presences of complex- and hybrid-type glycans, with galactose substituted with N-acetylgalactosamine. This substitution is typical for glycoproteins expressed in the bovine mammary gland. A specific ELISA revealed bNGAL concentrations in plasma and mature milk of about 0.05 and 1 microg/mL, respectively, whereas values as high as 51 microg/mL were measured in colostrum. Thus, we have isolated and characterized a novel bovine (milk) protein that is a new member of the lipocalin family.
van Veen
H A
HA
Pharming, Archimedesweg 4, 2333 CN Leiden, The Netherlands. h.veen@pharming.com
Geerts
M E J
ME
Zoetemelk
R A A
RA
Nuijens
J H
JH
van Berkel
P H C
PH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002417:Cattle / Q000502:physiology*; D002852:Chromatography, Ion Exchange / Q000662:veterinary; D003126:Colostrum / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary; D005260:Female; D018093:Gelatinases / Q000378:metabolism; D007962:Leukocytes / Q000737:chemistry; D054834:Lipocalins / Q000737:chemistry* / Q000302:isolation & purification; D009005:Monosaccharides / Q000737:chemistry; D009504:Neutrophils / Q000737:chemistry* / Q000201:enzymology; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods / Q000662:veterinary
D000906:Antibodies; D054834:Lipocalins; D009005:Monosaccharides; D018093:Gelatinases