Abstract
HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2W208F, HET-C2W208A and HET-C2F149Y all retained > 90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas HET-C2F149A transfer activity decreased to ~ 55% but displayed ~ 120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~ 85–90%) originates from Trp109. This conclusion was supported by HET-C2W109Y/F149Y which displayed ~ 8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λmax by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~ 30–40%) and λmax blue-shifts (~ 12 nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λmax blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λmax blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.
Original language | English (US) |
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Pages (from-to) | 1069-1076 |
Number of pages | 8 |
Journal | Biochimica et Biophysica Acta - Biomembranes |
Volume | 1860 |
Issue number | 5 |
DOIs | |
State | Published - May 2018 |
Bibliographical note
Funding Information:We are grateful for support from Dept. of Science and Technology, Science and Engineering Research Board (SERB), Govt. of India to Ravi Kanth Kamlekar ( YSS/2014/000021 ) and to Roopa Kenoth (YSS/2015/000783) as well as support by NIH / NIGMS-GM45928 (to REB), NIH/ NCI-CA121493 (to DJP & REB), NIH/ NHLBI-HL125353 (to DJP & REB) and the Hormel Foundation . We thank VIT for research facilities and infrastructure. Appendix A
Publisher Copyright:
© 2018 Elsevier B.V.
Keywords
- Fluorescence
- GLTP
- GLTP-fold
- Glycosphingolipid transfer
- HET-C2
- Membrane binding
- Trp point mutation