Related Experiment Videos
Mapping the heparin-binding site of mucus proteinase inhibitor
P Mellet1, J Ermolieff, J G Bieth
1Laboratoire d'Enzymologie, INSERM Unité 392, Université Louis Pasteur de Strasbourg, Illkirch, France.
Abstract:
Heparin accelerates the inhibition of neutrophil elastase by mucus proteinase inhibitor (MPI), the physiological antielastase of airways as a result of its binding with the inhibitor [Faller, B., Mély, Y., Gérard, D., & Bieth, J. G. (1992) Biochemistry 31, 8285-8290]. To explore the heparin-binding site of the inhibitor, we have modified the lysine and arginine residues of MPI and its isolated C-terminal domain by using 4-N,N-(dimethylamino)azobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC) [Chang, J. Y. (1989) J. Biol. Chem. 264, 3111-3115] and (p-hydroxyphenyl)glyoxal (HPG) (Yamasaki, R. B., Vega, A., & Feeney, R. E. (1980) Anal. Biochem. 109, 32-40], respectively. The derivatizations were done in the absence and presence of a 4.5 kDa heparin fraction with a low degree of polydispersity. The effect of chemical modification of the inhibitors on their affinity for heparin was tested using two complementary procedures, one based on the ability of heparin to accelerate the inhibition of chymotrypsin by the inhibitors and the other exploiting the affinity of the inhibitors for immobilized heparin. Modification of a limited number of lysine and arginine residues in full-length MPI led to a 6-fold decrease in affinity for heparin. The presence of the polymer during the modification reactions significantly prevented this effect. Amino acid sequencing unambiguously identified the heparin-protected lysines as Lys 13 and Lys 87, located on the N-terminal and C-terminal domains of MPI, respectively. Heparin apparently protects mainly two arginine residues from modification by HPG.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Heparin binding to mucus proteinase inhibitor (MPI) involves specific lysine residues. Modifying these sites significantly reduces heparin affinity, highlighting their crucial role in this interaction.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Mucus proteinase inhibitor (MPI) is the primary antielastase in airways.
- Heparin enhances MPI's inhibition of neutrophil elastase by binding to MPI.
- Understanding the heparin-binding site on MPI is crucial for elucidating this interaction.
Purpose of the Study:
- To identify the specific residues on MPI responsible for heparin binding.
- To investigate the role of lysine and arginine residues in heparin-MPI interaction.
- To determine if heparin protects specific residues during chemical modification.
Main Methods:
- Chemical modification of lysine and arginine residues in MPI and its C-terminal domain using S-DABITC and HPG.
- Derivatization performed in the absence and presence of a heparin fraction.
- Assessing heparin-MPI affinity using heparin-accelerated chymotrypsin inhibition and immobilized heparin assays.
- Amino acid sequencing to identify modified residues.
Main Results:
- Modification of a few lysine and arginine residues decreased MPI's heparin affinity sixfold.
- Heparin presence during modification significantly reduced the loss of affinity.
- Lysine residues 13 and 87 were identified as key heparin-protected sites.
- Heparin also appears to protect specific arginine residues from HPG modification.
Conclusions:
- Lysine residues 13 and 87 are critical for heparin binding to MPI.
- Heparin's interaction with MPI involves these specific lysine residues.
- The findings provide insights into the molecular basis of heparin-MPI interaction in airway physiology.