Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp.
BACKGROUND
The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction.


RESULTS
The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr.


CONCLUSIONS
Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.
Portugal
Michelle I
MI
Laboratório de Microbiologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, Av, Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21045-900, Brazil. michelleip@ig.com.br
Todeschini
Adriane R
AR
de Lima
Cristiana S
CS
Silva
Carlos A M
CA
Mohana-Borges
Ronaldo
R
Ottenhoff
Tom H M
TH
Mendonça-Previato
Lucia
L
Previato
Jose O
JO
Pessolani
Maria C V
MC
D018829:Adhesins, Bacterial / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009166:Mycobacterium leprae / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism
D018829:Adhesins, Bacterial; D006493:Heparin; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D012685:Sepharose; D012965:Sodium Chloride; C037405:heparin-sepharose