The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL.
Detection of matrix metalloproteinase (MMP) activities in the urine from patients with a variety of cancers has been closely correlated to disease status. Among these activities, the presence of a group of high molecular weight (HMW) MMPs independently serves as a multivariate predictor of the metastatic phenotype (). The identity of these HMW MMP activities has remained unknown despite their novelty and their potentially important applications in non-invasive cancer diagnosis and/or prognosis. Here, we report the identification of one of these HMW urinary MMPs of approximately 125-kDa as being a complex of gelatinase B (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL). Multiple biochemical approaches verified this identity. Analysis using substrate gel electrophoresis demonstrated that the 125-kDa urinary MMP activity co-migrates with purified human neutrophil MMP-9 x NGAL complex. The 125-kDa urinary MMP-9 x NGAL complex was recognized by a purified antibody against human NGAL as well as by a monospecific anti-human MMP-9 antibody. Furthermore, these same two antibodies were independently capable of specifically immunoprecipitating the 125-kDa urinary MMP activity in a dose-dependent manner. In addition, the complex of MMP-9 x NGAL could be reconstituted in vitro by mixing MMP-9 and NGAL in gelatinase buffers with pH values in the range of urine and in normal urine as well. Finally, the biochemical consequences of the NGAL and MMP-9 interaction were investigated both in vitro using recombinant human NGAL and MMP-9 and in cell culture by overexpressing NGAL in human breast carcinoma cells. Our data demonstrate that NGAL is capable of protecting MMP-9 from degradation in a dose-dependent manner and thereby preserving MMP-9 enzymatic activity. In summary, this study identifies the 125-kDa urinary gelatinase as being a complex of MMP-9 and NGAL and provides evidence that NGAL modulates MMP-9 activity by protecting it from degradation.
Yan
L
L
Department of Surgery, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Borregaard
N
N
Kjeldsen
L
L
Moses
M A
MA
D000209:Acute-Phase Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology; D004795:Enzyme Stability; D006801:Humans; D000071068:Lipocalin-2; D054834:Lipocalins; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism* / Q000652:urine; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology; D015513:Oncogene Proteins; D011233:Precipitin Tests; D020011:Protective Agents / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D014407:Tumor Cells, Cultured; D014551:Urinary Tract / Q000201:enzymology*
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D015513:Oncogene Proteins; D020011:Protective Agents; D011518:Proto-Oncogene Proteins; D020780:Matrix Metalloproteinase 9