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Proposed lipocalin fold for apolipoprotein M based on bioinformatics and site-directed mutagenesis
J Duan1, B Dahlbäck, B O Villoutreix
1Center for Structural Biochemistry, Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden. jianxin.duan@csb.ki.se
Abstract:
Apolipoprotein M (apoM) is a novel apolipoprotein that is predominantly present in high-density lipoprotein. Sensitive sequence searches, threading and comparative model building experiments revealed apoM to be structurally related to the lipocalin protein family. In a 3D model, characterized by an eight-stranded anti-parallel beta-barrel, a segment including Asn135 could adopt a closed or open conformation. Using site-directed mutagenesis, we demonstrated Asn135 in wild-type apoM to be glycosylated, suggesting that the segment is solvent exposed. ApoM displays two strong acidic patches of potential functional importance, one around the N-terminus and the other next to the opening of the beta-barrel.
Insights
Apolipoprotein M (apoM), found in high-density lipoprotein, is structurally similar to lipocalins. We found that Asn135 in apoM is glycosylated, indicating a solvent-exposed region potentially involved in function.
Area of Science:
- Biochemistry
- Structural Biology
- Lipid Metabolism
Background:
- Apolipoprotein M (apoM) is a protein primarily found in high-density lipoprotein (HDL).
- Its structural characteristics and functional roles are not fully elucidated.
- Understanding apoM structure is crucial for its role in lipid transport and metabolism.
Purpose of the Study:
- To investigate the structural relationship of apolipoprotein M to known protein families.
- To identify and characterize post-translational modifications and functional regions of apoM.
- To build a 3D model of apoM for structural and functional insights.
Main Methods:
- Sequence searches, threading, and comparative model building were used to determine apoM's structural family.
- Site-directed mutagenesis was employed to investigate specific amino acid residues.
- Glycosylation analysis was performed to identify post-translational modifications.
Main Results:
- Apolipoprotein M shares structural similarities with the lipocalin protein family, featuring a beta-barrel structure.
- A specific segment around Asn135 was identified, capable of adopting open or closed conformations.
- Site-directed mutagenesis confirmed that Asn135 in wild-type apoM is glycosylated, suggesting solvent exposure.
- Two acidic patches, at the N-terminus and near the beta-barrel opening, were identified as potentially important functional sites.
Conclusions:
- Apolipoprotein M is a lipocalin family member with a distinct beta-barrel structure.
- The glycosylation of Asn135 indicates a solvent-exposed region important for apoM function.
- The identified acidic patches may play a role in apoM's interactions and functions within HDL.