Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein

Roopa Kenoth, Xianqiong Zou, Dhirendra K. Simanshu, Helen M. Pike, Lucy Malinina, Dinshaw J. Patel, Rhoderick E. Brown, Ravi Kanth Kamlekar

Research output: Contribution to journalArticle

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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 languageEnglish (US)
Pages (from-to)1069-1076
Number of pages8
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1860
Issue number5
DOIs
StatePublished - May 1 2018

Fingerprint

Glycolipids
Tryptophan
Membranes
Neutral Glycosphingolipids
Fluorescence
Phosphorylcholine
Sugars
Ethanol
Lipids
Injections
lipid transfer protein

Keywords

  • Fluorescence
  • GLTP
  • GLTP-fold
  • Glycosphingolipid transfer
  • HET-C2
  • Membrane binding
  • Trp point mutation

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

Cite this

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein. / Kenoth, Roopa; Zou, Xianqiong; Simanshu, Dhirendra K.; Pike, Helen M.; Malinina, Lucy; Patel, Dinshaw J.; Brown, Rhoderick E.; Kamlekar, Ravi Kanth.

In: Biochimica et Biophysica Acta - Biomembranes, Vol. 1860, No. 5, 01.05.2018, p. 1069-1076.

Research output: Contribution to journalArticle

Kenoth, Roopa ; Zou, Xianqiong ; Simanshu, Dhirendra K. ; Pike, Helen M. ; Malinina, Lucy ; Patel, Dinshaw J. ; Brown, Rhoderick E. ; Kamlekar, Ravi Kanth. / Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein. In: Biochimica et Biophysica Acta - Biomembranes. 2018 ; Vol. 1860, No. 5. pp. 1069-1076.
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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.",
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AU - Kenoth, Roopa

AU - Zou, Xianqiong

AU - Simanshu, Dhirendra K.

AU - Pike, Helen M.

AU - Malinina, Lucy

AU - Patel, Dinshaw J.

AU - Brown, Rhoderick E.

AU - Kamlekar, Ravi Kanth

PY - 2018/5/1

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N2 - 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.

AB - 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.

KW - Fluorescence

KW - GLTP

KW - GLTP-fold

KW - Glycosphingolipid transfer

KW - HET-C2

KW - Membrane binding

KW - Trp point mutation

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