Purification and Validation of Lipid Transfer Proteins

Kjellberg, Matti A.; Backman, Anders P. E.; Möuts, Anna; Mattjus, Peter

doi:10.1007/978-1-4939-6996-8_19

Cited by 3 publications

(4 citation statements)

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“…We further examined the ability of GLTP to recognize and transfer GlcCer of different lengths using an indirect method previously developed for analyzing the substrate specificity of lipid transfer proteins [27, 36]. Since GLTP does not transfer sphingomyelin, the transfer of TopFluor-GlcCer by GLTP was unaffected by the addition of palmitoyl-sphingomyelin (PSM) as a competitor.…”

Section: Resultsmentioning

confidence: 99%

“…Small differences in the buffer conditions result in large variations in the SPR response. Therefore, using the same buffer throughout all experimental steps is important, as well as the final steps in the protein purification procedure [27]. The protein solution was filtered (0.2 μm) and stored at 4°C for a maximum of 7 days.…”

Section: Methodsmentioning

confidence: 99%

“…The transfer assay was started by addition of 0.25–2 μg of GLTP. The transfer rates during the first minute after GLTP injection, termed the initial transfer rate, can be calculated by determining the increase in the fluorescence intensity (BODIPY-GlcCer unquenching) and comparing this value to the total fluorescence intensity obtained after adding Triton X-100 (final concentration 1%) and subtracting a Triton X-100 blank [27]. Each experiment (donor vesicle preparation) was repeated at least three times, and at least three transfer measurements were conducted per experiment.…”

Section: Methodsmentioning

confidence: 99%

“…Unlabeled GlcCer (1 mol%) was added to the donor vesicles composed of either POPC or DOPC, as described above. The addition of an unlabeled glycosphingolipid would result in a decrease in the transfer rate of the fluorescently labeled lipid, if the added GlcCer was competing with the TopFluor-GlcCer [27, 36]. If the added lipids are not substrates for the transfer protein, no deviation in the slope of the transfer rate for TopFluor-GlcCer would occur.…”

Section: Methodsmentioning

confidence: 99%

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Glucosylceramide acyl chain length is sensed by the glycolipid transfer protein

et al. 2018

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The glycolipid transfer protein, GLTP, can be found in the cytoplasm, and it has a FFAT-like motif (two phenylalanines in an acidic tract) that targets it to the endoplasmic reticulum (ER). We have previously shown that GLTP can bind to a transmembrane ER protein, vesicle-associated membrane protein-associated protein A (VAP-A), which is involved in a wide range of ER functions. We have addressed the mechanisms that might regulate the association between GLTP and the VAP proteins by studying the capacity of GLTP to recognize different N-linked acyl chain species of glucosylceramide. We used surface plasmon resonance and a lipid transfer competition assay to show that GLTP prefers shorter N-linked fully saturated acyl chain glucosylceramides, such as C8, C12, and C16, whereas long C18, C20, and C24-glucosylceramides are all bound more weakly and transported more slowly than their shorter counterparts. Changes in the intrinsic GLTP tryptophan fluorescence blueshifts, also indicate a break-point between C16- and C18-glucosylceramide in the GLTP sensing ability. It has long been postulated that GLTP would be a sensor in the sphingolipid synthesis machinery, but how this mechanistically occurs has not been addressed before. It is unclear what proteins the GLTP VAP association would influence. Here we found that if GLTP has a bound GlcCer the association with VAP-A is weaker. We have also used a formula for identifying putative FFAT-domains, and we identified several potential VAP-interactors within the ceramide and sphingolipid synthesis pathways that could be candidates for regulation by GLTP.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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