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KanagaVijayan D, Subramanian R, Santhakumari PR, et al. Structure of recombinantly expressed cockroach Lili-Mip protein in glycosylated and deglycosylated forms. Biochim Biophys Acta Gen Subj. 2021;1866(3):130064doi: 10.1016/j.bbagen.2021.130064.
KanagaVijayan, D., Subramanian, R., Santhakumari, P. R., Chavas, L. M. G., Subramanian, R., & Banerjee, S. (2021). Structure of recombinantly expressed cockroach Lili-Mip protein in glycosylated and deglycosylated forms. Biochimica et biophysica acta. General subjects, 1866(3), 130064. https://doi.org/10.1016/j.bbagen.2021.130064
KanagaVijayan, Dhanabalan, et al. "Structure of recombinantly expressed cockroach Lili-Mip protein in glycosylated and deglycosylated forms." Biochimica et biophysica acta. General subjects vol. 1866,3 (2021): 130064. doi: https://doi.org/10.1016/j.bbagen.2021.130064
KanagaVijayan D, Subramanian R, Santhakumari PR, Chavas LMG, Subramanian R, Banerjee S. Structure of recombinantly expressed cockroach Lili-Mip protein in glycosylated and deglycosylated forms. Biochim Biophys Acta Gen Subj. 2021 Dec 24;1866(3):130064. doi: 10.1016/j.bbagen.2021.130064. Epub 2021 Dec 24. PMID: 34958847.
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Biochim Biophys Acta Gen Subj. 2021 Dec 24;1866(3):130064. doi: 10.1016/j.bbagen.2021.130064. Epub 2021 Dec 24.

Structure of recombinantly expressed cockroach Lili-Mip protein in glycosylated and deglycosylated forms.

Biochimica et biophysica acta. General subjects

Dhanabalan KanagaVijayan, Rudra Subramanian, Partha Radhakrishnan Santhakumari, Leonard M G Chavas, Ramaswamy Subramanian, Sanchari Banerjee

Affiliations

  1. Biological Sciences, Purdue University, West Lafayette, IN 47907, USA; Institute for Stem Cell Science and Regenerative Medicine, Bengaluru, Karnataka 560065, India.
  2. Bindley Biosciences Centre, Purdue University, West Lafayette, IN 47906, USA.
  3. Biological Sciences, Purdue University, West Lafayette, IN 47907, USA; Institute for Stem Cell Science and Regenerative Medicine, Bengaluru, Karnataka 560065, India; Manipal Academy of Higher Education, Tiger Circle Road, Manipal, Karnataka 576104, India.
  4. Synchrotron Radiation Research Center, Nagoya University, Nagoya 4648603, Japan.
  5. Biological Sciences, Purdue University, West Lafayette, IN 47907, USA; Bindley Biosciences Centre, Purdue University, West Lafayette, IN 47906, USA; Institute for Stem Cell Science and Regenerative Medicine, Bengaluru, Karnataka 560065, India. Electronic address: [email protected].
  6. Institute for Stem Cell Science and Regenerative Medicine, Bengaluru, Karnataka 560065, India; Department of Chemistry, University of Copenhagen, Universitetsparken 5, Copenhagen 2100, Denmark.

PMID: 34958847 DOI: 10.1016/j.bbagen.2021.130064

Abstract

BACKGROUND: The Pacific Beetle Cockroach is the only known viviparous cockroach. The pregnant females provide nutrition to the embryos by secreting milk proteins (Lili-Mips), which crystallize in vivo. The crystals that grow in the embryo are heterogeneous in their protein sequence. It is not apparent from the structure determined what role heterogeneity and glycosylation played in crystallization. Lili-Mips are very nutritious.

METHODS: Here, we report the cloning of synthesized Lili-Mip genes, their expression in Saccharomyces cerevisiae as secreted proteins, purification, crystallization, and the determination of a three-dimensional structure of one glycosylated and one deglycosylated form.

RESULTS: A 2.35 Å structure of the glycosylated form is bound to palmitoleic acid and has several Zn atom mediated interactions. A 1.45 Å structure of the deglycosylated protein reveals a binding pocket that has both oleic and palmitoleic acid bound. Mass-spectrometry shows that oleic acid and palmitoleic acid are bound to the protein. Docking studies suggest that aliphatic chains of lengths 15, 16, and 18 carbons bind well in the pocket.

CONCLUSIONS: The recombinantly expressed and secreted protein is glycosylated, has a bound fatty acid, is homogenous in its protein sequences, and readily forms crystals. The deglycosylated protein also crystallizes readily, suggesting that the high crystallizability of this protein is independent of glycosylation.

GENERAL SIGNIFICANCE: Lili-Mips belong to the ubiquitous lipocalin family of proteins that bind to a large variety of ligands. While the residues lining the barrel are essential for the affinity of the ligand, our results show the role of side-chain orientations to ligand selectivity.

Copyright © 2021 Elsevier B.V. All rights reserved.

Keywords: Diploptera punctata; Glycosylation; Lipocalin-like Milk proteins; Oleic acid (OLA); Palmitoleic acid (PAM); Saccharomyces cerevisiae

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