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The disulfide bond arrangement of leukemia inhibitory factor: homology to oncostatin M and structural implications
N A Nicola1, B Cross, R J Simpson
1Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia.
Abstract:
Murine leukemia inhibitory factor (LIF) (the fully active recombinant form produced in E. coli) was digested in the unreduced state with trypsin and Staphylococcal V8 protease in 0.05% sodium dodecyl sulfate. Disulfide-bonded peptides were identified by altered mobility on reverse-phase high-performance liquid chromatography in the presence or absence of dithiothreitol and subjected to amino acid sequencing. Peptides containing more than one disulfide bond were subjected to further proteolysis and disulfide-bonded subfragments identified and sequenced. The three disulfide bonds are CYS13-CYS135, CYS19-CYS132 and CYS61-164 and the first and third of these are clearly homologous to the two disulfide bonds in oncostatin M. The spatial organization of the cysteine residues in the predicted four alpha-helical bundle structure of LIF (Bazan, Neuron 7,197;1991) is compatible with these disulfide assignments.
Insights
Researchers mapped the disulfide bonds in murine leukemia inhibitory factor (LIF), a key protein. This structural information is crucial for understanding LIF
Area of Science:
- Biochemistry
- Proteomics
- Structural Biology
Background:
- Murine leukemia inhibitory factor (LIF) is a secreted glycoprotein with critical roles in cell proliferation, differentiation, and survival.
- Understanding the protein's three-dimensional structure is essential for elucidating its biological functions and interactions.
- Disulfide bonds play a vital role in protein folding and stability.
Purpose of the Study:
- To determine the precise locations of the disulfide bonds within recombinant murine leukemia inhibitory factor (LIF).
- To provide insights into the structural organization of LIF and its relationship to homologous proteins like oncostatin M.
Main Methods:
- Proteolytic digestion of recombinant murine leukemia inhibitory factor (LIF) using trypsin and Staphylococcal V8 protease.
- Identification of disulfide-bonded peptides via reverse-phase high-performance liquid chromatography (RP-HPLC) with and without dithiothreitol (DTT).
- Amino acid sequencing of identified peptides and subfragments to confirm disulfide bond linkages.
Main Results:
- Three disulfide bonds were identified in murine leukemia inhibitory factor (LIF): CYS13-CYS135, CYS19-CYS132, and CYS61-164.
- The first (CYS13-CYS135) and third (CYS61-164) disulfide bonds show homology to those found in oncostatin M.
- The determined disulfide bond arrangement is consistent with the predicted alpha-helical bundle structure of LIF.
Conclusions:
- The study precisely mapped the three disulfide bonds in murine leukemia inhibitory factor (LIF).
- These findings support the proposed structural model of LIF and highlight conserved structural features with oncostatin M.
- This detailed structural information is fundamental for future research on LIF function and therapeutic development.