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Identification of receptors for iron-binding protein structures

Vibeke Hvidberg  


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Accepted by: Faculty of Health Sciences University of Aarhus
Defended on: March 14, 2008
Official opponents: Steffen Thiel, PhD, Associate Professor , Timo K. van den Berg, PhD, Associate Professor , Emanuela Tolosano, PhD, Associate Professor
Tutors: Søren Kragh Moestrup, dr.med., Professor

Published in the PhD Database: May 7, 2008


English abstract
The PhD thesis ¿Identification of receptors for iron-binding protein structures¿ is based on experimental work performed at the Department of Medical Biochemistry, University of Aarhus. The work was conducted to identify the endocytic receptors involved in hemopexin (Hx)-heme and neutrophil-gelatinase-associated lipocalin (NGAL)-iron-siderophore uptake. When these receptors were identified, analyses were performed to establish whether they also function as receptors for the gelatinase B/MMP9 enzyme as MMP9 is associated with NGAL in neutrophils and contains a Hx domain possibly involved in receptor recognition. The main results are presented in three scientific papers.
Approximately 70% of body iron is incorporated into hemoglobin in circulating erythrocytes. This iron is recycled through the breakdown of senescent erythrocytes by the macrophages of the reticuloendothelial system. Normally, intravascular hemolysis accounts for around 10% of erythrocyte breakdown but it is highly elevated in pathological conditions such as malaria, thalassemias, and sickle cell disease. Hemoglobin in plasma may dissociate into free heme and globin. The toxic free heme is rapidly sequestered by the circulating serum protein Hx which transports heme to the liver for breakdown and reutilization of the iron atom.
Another secondary iron assimilation strategy is conducted by NGAL which is released from activated neutrophils at sites of infection. NGAL binds iron-siderophore complexes thereby depriving invading pathogens of iron. In order to sustain this antimicrobial effect, host internalization of NGAL-iron-siderophore complexes would be beneficial.
In the first study published in Blood, the low density lipoprotein (LDL) receptor-related protein (LRP)/CD91 was identified as the long-sought Hx-heme receptor by affinity chromatography. Binding studies including surface plasmon resonance (SPR) analysis confirmed that LRP/CD91 binding of Hx-heme is specific and dependent on Hx-heme complex formation. Analysis of cellular LRP/CD91-mediated endocytosis of Hx-heme revealed that Hx is degraded in lysosomes. This was an unexpected finding as recycling of Hx back into the circulation has previously been hypothesized. In addition, real-time PCR analysis of mRNA levels of the heme-degrading enzyme heme oxygenase-1 (HO-1) showed that LRP/CD91-mediated uptake of Hx-heme complexes results in induction of HO-1 mRNA.
The second study published in FEBS Letters reports the identification of megalin, another member of the LDL receptor family, as a receptor for the iron-siderophore-binding lipocalin NGAL. By SPR analysis, megalin was shown to bind both apoNGAL and NGAL-iron-siderophore complexes with equal affinities. Megalin-dependent NGAL uptake was verified in cellular uptake studies.
The third report published in Journal of Biological Chemistry concerned the gelatinase B/MMP9 enzyme known to be internalized via LRP/CD91. Megalin was identified as a receptor for MMP9. SPR analysis of the binding of recombinant MMP9 variants to LRP/CD91 and megalin showed that receptor binding is mediated by the MMP9 Hx domain while the highly O-glycosylated MMP9 linker domain ensures the correct positioning of the Hx domain for optimal receptor recognition.
In conclusion, this PhD study has provided new information on novel players and the mechanisms involved in regulation of iron and MMP9 metabolism.



Danish abstract
Det eksperimentelle arbejde, der danner grundlag for denne ph.d.-afhandling med titlen ¿ Identifikation af receptorer for jernbindende protein strukturer¿, er udført ved Institut for Medicinsk Biokemi, Aarhus Universitet. Formålet var at identificere receptorerne for det hæm-bindende serum protein hæmopexin og det jern-bindende protein neutrofil-gelatinase-associeret lipocalin (NGAL). Derudover blev interaktionen mellem matrix metalloproteinase-9 (MMP9) og dets receptorer low density lipoprotein receptor-related protein (LRP) og megalin analyseret. Resultaterne af disse undersøgelser er præsenteret i tre separate videnskabelige rapporter.
Den første rapport omhandler identifikationen af LRP som receptoren, der endocyterer hæm-hæmopexin komplekset i primært leveren. LRP blev isoleret fra cellemembraner fra human placenta og lever ved hæm-hæmopexin affinitetskromatografi. Efterfølgende blev interaktionen mellem hæm-hæmopexin komplekset og LRP bekræftet ved surface plasmon resonance analyse. Disse forsøg viste også, at apohæmopexin ikke binder LRP. At cellulær optagelse af hæm-hæmopexin komplekser specifikt varetages af LRP blev vist i forsøg med cellekulturer. Endvidere blev det vist, at LRP-medieret endocytose af hæm-hæmopexin medfører lysosomal nedbrydning af hæmopexin samt inducerer transkription af hæm oxygenase-1. Resultaterne er publiceret i tidsskriftet Blood.
Den anden rapport viser, at megalin er receptor for både apoNGAL og NGAL i kompleks med jern og bakterielle siderophorer. Dette blev påvist ved surface plasmon resonance analyse samt ved endocytose-forsøg i cellekulturer. Resultaterne er publiceret i tidsskriftet FEBS Letters.
Den tredje rapport omhandler MMP9 og dets interaktion med LRP og megalin. LRP er tidligere identificeret som receptor for MMP9, mens megalin blev identificeret som sådan i dette studium vha. surface plasmon resonance analyse og endocytose-forsøg i cellekulturer. Forskellige deletionsmutanter af MMP9 blev udtrykt i insektceller og analyseret for binding til LRP og megalin ved surface plasmon resonance analyse. Disse forsøg viste, at LRP og megalin binder til MMP9¿s hæmopexin domæne. Resultaterne er publiceret i tidsskriftet Journal of Biological Chemistry.
Denne ph.d.-afhandling har således bidraget med ny viden om jernstofskiftet især i forbindelse med intravaskulær hæmolyse og bakterielle infektioner, idet hæmopexins og NGAL¿s primære funktioner er sekvestrering af henholdsvis hæm og jern i disse tilstande. Identifikationen af de involverede receptorer muliggør nærmere undersøgelser af jernmetabolismen. Ydermere bidrager karakteriseringen af interaktionen mellem MMP9 og LRP og megalin til en større forståelse af MMP9 regulering og dermed aktivitet f.eks. i forbindelse med tumor progression.