Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
1	bDGlcp	103.78	73.62	76.71	70.29	77.15	61.34
2	lR2HOC18={t3}
	xXSR9b1SphdC19


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
1	bDGlcp	4.128	2.967	3.150	3.047	3.101	3.441-3.667
2	lR2HOC18={t3}
	xXSR9b1SphdC19


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
1	bDGlcp	103.78/4.128	73.62/2.967	76.71/3.150	70.29/3.047	77.15/3.101	61.34/3.441-3.667
2	lR2HOC18={t3}
	xXSR9b1SphdC19

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CCCCCCCCCCCCCC/C=C/[C@@H](O)C(=O)N[C@@H](CO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O)[C@H](O)/C=C/CC/C=C(\C)CCCCCCCCC

754.103 g/mol (C₄₃H₇₉NO₉)

Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
1	bDGalp	104.43	70.80	73.54	68.35	75.58	60.65
2	lR2HOC18={t3}
	xXSR9b1SphdC19


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
1	bDGalp	4.079	3.296	3.288	3.631	3.332	3.480-3.531
2	lR2HOC18={t3}
	xXSR9b1SphdC19


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
1	bDGalp	104.43/4.079	70.80/3.296	73.54/3.288	68.35/3.631	75.58/3.332	60.65/3.480-3.531
2	lR2HOC18={t3}
	xXSR9b1SphdC19

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CCCCCCCCCCCCCC/C=C/[C@@H](O)C(=O)N[C@@H](CO[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O)[C@H](O)/C=C/CC/C=C(\C)CCCCCCCCC

754.103 g/mol (C₄₃H₇₉NO₉)

Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
1	bDGlcp	103.78	73.62	76.71	70.29	77.15	61.34
2	lR2HOSte
	xXSR9b1SphdC19


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
1	bDGlcp	4.127	2.963	3.147	3.042	3.099	3.436-3.667
2	lR2HOSte
	xXSR9b1SphdC19


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
1	bDGlcp	103.78/4.127	73.62/2.963	76.71/3.147	70.29/3.042	77.15/3.099	61.34/3.436-3.667
2	lR2HOSte
	xXSR9b1SphdC19

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CCCCCCCCCCCCCCCC[C@@H](O)C(=O)N[C@@H](CO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O)[C@H](O)/C=C/CC/C=C(\C)CCCCCCCCC

756.119 g/mol (C₄₃H₈₁NO₉)