Journal: The Journal of Biological Chemistry

Article Title: Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides

doi: 10.1016/j.jbc.2023.104603

Figure Lengend Snippet: Greatly reduced but residual presence of PHS-containing CERs in the epidermis of Degs2 KO mice. Lipids were extracted from epidermis of postnatal day 0 WT (n = 3) and Degs2 KO (n = 3) mice, and CERs ( A and B ) and acyl-CERs ( C and D ) were measured via LC-MS/MS. Values presented are means + SD (∗∗ p < 0.01; Student’s t test) of the total quantities of PHS-CERs, SPH-CERs, and DHS-CERs ( A ); the quantity of each PHS-CER species containing the indicated FA moiety ( B ); the total quantities of acyl-PHS-CERs, acyl-SPH-CERs, and acyl-DHS-CERs ( C ); and the quantity of each acyl-PHS-CER species containing the indicated ω-hydroxy FA ( D ). CER, ceramide; DHS-CER, DHS containing CER; FA, fatty acid; PHS-CER, PHS-containing CER; SPH-CER, SPH-containing CER.

Article Snippet: To these samples, we added 375 μl of chloroform/methanol (1:2, v/v) and d 9 -labeled CER/acyl-CER standards ( d 9 -SPH-CER, 10 pmol; d 9 -DHS-CER, 5 pmol; d 9 -PHS-CER, 2.5 pmol; N -(26-oleoyloxy( d 9 ) hexacosanoyl) D- erythro -SPH [ d 9 -acyl-SPH-CER], 10 pmol [Avanti Polar Lipids]) and mixed vigorously.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides

doi: 10.1016/j.jbc.2023.104603

Figure Lengend Snippet: Greatly reduced but residual presence of PHS-containing CERs in DEGS2 KO keratinocytes. A , DEGS2 KO keratinocytes were generated using the CRISPR/Cas9 system. The exon structure ( black , coding sequence; white , untranslated regions) of human DEGS2 and the nucleotide sequences of WT and DEGS2 KO keratinocytes (KO clones 1 and 2) around the guide RNA target sequences ( light blue ) and the protospacer-adjacent motif sequences ( magenta ) in exon 2 are shown. B – D , lipids were extracted from controls (controls 1 and 2) and DEGS2 KO keratinocytes (KO 1 and 2) differentiated for 14 days, and CERs and acyl-CERs were analyzed via LC-MS/MS. Values presented are means + SD (n = 3; ∗∗ p < 0.01; ∗ p < 0.05; Scheffé’s test) of the total quantities of PHS-CERs, SPH-CERs, DHS-CERs, acyl-PHS-CERs, acyl-SPH-CERs, and acyl-DHS-CERs ( B ); the quantity of each PHS-CER species containing the indicated FA moiety ( C ); and the quantity of each acyl-PHS-CER species containing the indicated ω-hydroxy FA ( D ). E , HEK 293T cells were transfected with pCE-puro 3× FLAG-1 (vector) or pCE-puro 3× FLAG-FA2H plasmid. After 24 h of transfection, lipids were extracted, and 2-hydroxy palmitic acid-containing SPH-CER (2-OH CER) and PHS-CERs were quantified via LC-MS/MS. Values presented are means + SD (n = 3; ∗∗ p < 0.01; ∗ p < 0.05; Student’s t test). CER, ceramide; DHS-CER, DHS containing CER; PHS-CER, PHS-containing CER; SPH-CER, SPH-containing CER.

Article Snippet: To these samples, we added 375 μl of chloroform/methanol (1:2, v/v) and d 9 -labeled CER/acyl-CER standards ( d 9 -SPH-CER, 10 pmol; d 9 -DHS-CER, 5 pmol; d 9 -PHS-CER, 2.5 pmol; N -(26-oleoyloxy( d 9 ) hexacosanoyl) D- erythro -SPH [ d 9 -acyl-SPH-CER], 10 pmol [Avanti Polar Lipids]) and mixed vigorously.

Techniques: Generated, CRISPR, Sequencing, Clone Assay, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides

doi: 10.1016/j.jbc.2023.104603

Figure Lengend Snippet: Preference for VLC substrates in the hydroxylation reaction by DEGS2. A and B , DEGS1 KO HAP1 cells were transfected with pEFh-3× FLAG-1 (vector), pEFh-3× FLAG-DEGS1, or pEFh-3× FLAG-DEGS2 plasmid. After 24 h, cells were labeled with 2 μM d 7 -DHS for 4 h. A , proteins were prepared and subjected to immunoblotting (IB) using anti-FLAG or anti-GAPDH (loading control) antibodies. B , lipids were extracted, and d 7 -labeled SPH-CERs, PHS-CERs, SPH-HexCERs, PHS-HexCERs, SPH-SMs, and PHS-SMs were quantified via LC-MS/MS. Values presented are means + SD of the respective lipids, with FA species color-coded (n = 3; ∗∗ p < 0.01; Dunnett’s test versus vector control). CER, ceramide; DHS-CER, DHS containing CER; FA, fatty acid; HexCER, monohexosylceramide; ND, not detected; PHS-CER, PHS-containing CER; SM, sphingomyelin; SPH-CER, SPH-containing CER.

Article Snippet: To these samples, we added 375 μl of chloroform/methanol (1:2, v/v) and d 9 -labeled CER/acyl-CER standards ( d 9 -SPH-CER, 10 pmol; d 9 -DHS-CER, 5 pmol; d 9 -PHS-CER, 2.5 pmol; N -(26-oleoyloxy( d 9 ) hexacosanoyl) D- erythro -SPH [ d 9 -acyl-SPH-CER], 10 pmol [Avanti Polar Lipids]) and mixed vigorously.

Techniques: Transfection, Plasmid Preparation, Labeling, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides

doi: 10.1016/j.jbc.2023.104603

Figure Lengend Snippet: Tissue distribution of t20:0 PHS-CERs and their production by DEGS2. A and B , lipids were extracted from six tissues (kidney, esophagus, anterior stomach, posterior stomach, small intestine, and large intestine) of 2-month-old WT mice (n = 3), and the quantities of SPH-CERs and PHS-CERs containing LCBs with chain lengths from d/t16 to d/t26 were quantified via LC-MS/MS. Values presented are means + SD of the total quantities of SPH-CERs and PHS-CERs ( A ; LCB chain lengths are color coded) and the quantities of t18:0- or t20:0-containing PHS-CERs ( B ; FA chain lengths are color-coded). C , lipids were extracted from the esophagus and anterior stomach of six-month-old WT (n = 3) and Degs2 KO (n = 3) mice, and the quantities of t18:0 and t20:0 PHS-CERs were examined via LC-MS/MS. Values presented are means + SD of the total quantities of PHS-CERs, with FA species color coded. D and E , esophagus and anterior stomach of 6-month-old WT and Degs2 KO mice were prepared and subjected to hematoxylin/eosin staining ( D ) and lucifer yellow assay ( E ). Scale bars, 50 μm ( D ) and 10 μm ( E ). F , lipids were extracted from the esophagus and anterior stomach of 6-month-old WT (n = 3) and Degs2 KO (n = 3) mice, and PHS-CERs, SPH-CERs containing 2-hydroxy FA (2-OH CERs), and SPH-CERs containing 3-hydroxy FA (3-OH CERs) were quantified via LC-MS/MS. Values presented are means + SD of the total quantities of PHS-CERs, 2-OH CERs, and 3-OH CERs. G and H , DEGS1 KO HAP1 cells were transfected with pCE-puro 3× FLAG-SPTLC1, pCE-puro 3× FLAG-SPTSSB, and pCE-puro HA-SPTLC3 plasmids together with pEFh-3× FLAG-1 (vector), pEFh-3× FLAG-DEGS1, or pEFh-3× FLAG-DEGS2 plasmid, and cultured for 24 h. G , proteins were prepared and subjected to immunoblotting (IB) with anti-FLAG, anti-HA, or anti-GAPDH (loading control) antibodies. H , lipids were extracted, and d20:1 SPH-CERs and t20:0 PHS-CERs were analyzed via LC-MS/MS. Values presented are means + SD of the total quantities of the respective lipids, with FA chain length color coded (n = 3; ∗∗ p < 0.01; Dunnett’s test versus vector control for d20:1 SPH-CERs and Student’s t test for t20:0 PHS-CERs). CER, ceramide; DHS-CER, DHS containing CER; FA, fatty acid; PHS-CER, PHS-containing CER; SPH-CER, SPH-containing CER.

Article Snippet: To these samples, we added 375 μl of chloroform/methanol (1:2, v/v) and d 9 -labeled CER/acyl-CER standards ( d 9 -SPH-CER, 10 pmol; d 9 -DHS-CER, 5 pmol; d 9 -PHS-CER, 2.5 pmol; N -(26-oleoyloxy( d 9 ) hexacosanoyl) D- erythro -SPH [ d 9 -acyl-SPH-CER], 10 pmol [Avanti Polar Lipids]) and mixed vigorously.

Techniques: Liquid Chromatography with Mass Spectroscopy, Staining, Transfection, Plasmid Preparation, Cell Culture, Western Blot

Journal: The Journal of Neuroscience

Article Title: Spiral Ganglion Degeneration and Hearing Loss as a Consequence of Satellite Cell Death in Saposin B-Deficient Mice

doi: 10.1523/JNEUROSCI.3920-13.2015

Figure Lengend Snippet: Liquid chromatography/mass spectrometry analysis of sulfatide levels in B−/− cochlear SGs. A, Significant alcian blue staining of the B−/− cochlear SGs (blue stain) is seen when compared with the WT cochlear SGs, indicative of sulfatide accumulation in SG neurons of B−/− mice. The whole cochlear section (top, low magnification; scale bar, 200 μm) and a higher magnification of the SGs (bottom; scale bar, 50 μm) are shown. B, Most of the NFA sulfatide (top) and HFA sulfatide (bottom) species were increased in B−/− mouse SGs compared with WT mouse SGs. Total NFA sulfatides and HFA sulfatides were increased in B−/− mouse SGs by 1.74-fold and 1.44-fold, respectively, when compared with that of WT mice SGs. C, C18 ceramide and total ceramides show no significant increase in the B−/− SGs when compared with that of WT SGs. D, Most NFA sulfatides are increased when normalized to C18 ceramide in the same sample. E, Most HFA sulfatide species are also significantly increased when normalized to C18 ceramide in the same sample. Total sulfatides in B−/− SGs were increased by 1.65-fold compared with WT SGs, mirroring the increased alcian blue staining on SGs in cochlea (A).

Article Snippet: Sulfatide and ceramide analysis using liquid chromatography/tandem mass spectrometry Synthetic N -acyl ceramide standards (C16, C17, C18, C24 Cer) of 99% purity were from Avanti Lipids.

Techniques: Liquid Chromatography, Mass Spectrometry, Staining

Journal: The Journal of Biological Chemistry

Article Title: Generation and validation of recombinant antibodies to study human aminoacyl-tRNA synthetases

doi: 10.1074/jbc.RA120.012893

Figure Lengend Snippet: List of aminoacyl-tRNA synthetases with antigen details Clinical names are used for descriptions of aaRS as autoantigens in idiopathic inflammatory myopathies. Antigen aa indicates the antigen amino acid coverage. Yield indicates the yield of purified antigen in mg/liter of E. coli culture as determined from absorbance at 280 nm after IMAC and SEC purification. The Addgene ID column shows the expression vectors of each of the constructs are available through Addgene accession numbers 153046–153060.

Article Snippet: Finally, the scFv are tested for their capacity to bind the endogenous target in cell lysate by IP-MS. Antibodies validated according to gold standard criteria are distributed via ULTRA-DD. table ft1 table-wrap mode="anchored" t5 caption a7 HUGO gene nomenclature committee name Protein name Clinical name NCBI Entrez Gene ID Target annotation Antigen aa Yield Vector Addgene ID mg/liter AARS AlaRS PL-12 16 Alanyl-tRNA synthetase Met 1 –Lys 455 38.7 pNIC-Bio3 153046 Met 1 –Asn 968 12.8 153047 DARS AspRS 1615 Aspartyl-tRNA synthetase Met 1 –Pro 501 37.3 pNIC-Bio3 153048 GARS GlyRS EJ 2617 Glycyl-tRNA synthetase Met 55 –Glu 739 16.4 pNIC-Bio3 153049 HARS HisRS Jo1 3035 Histidyl-tRNA synthetase Met 1 –Cys 509 55.6 pNIC-Bio3 153050 KARS LysRS 3735 Lysyl-tRNA synthetase Met 1 –V597 19.7 pNIC-Bio3 153051 LARS LeuRS 51520 Leucyl-tRNA synthetase Gly 260 –Glu 511 5.9 pNIC-Bio3 153052 MARS MetRS 4141 Methionyl-tRNA synthetase Met 1 –Leu 225 1.5 pNIC-CTB10H 153053 NARS AsnRS KS 4677 Asparaginyl-tRNA synthetase Met 1 –Pro 548 8.7 pNIC-Bio3 153054 RARS ArgRS 5917 Arginyl-tRNA synthetase Thr 70 –Met 660 17.7 pNIC-Bio3 153055 SARS SerRS 6301 Seryl-tRNA synthetase Met 1 –Ala 514 7.7 pNIC-CTB10H 153056 TARS ThrRS PL-7 6897 Threonyl-tRNA synthetase Asn 320 –Phe723 67.2 pNIC-Bio3 153057 WARS TrpRS 7453 Tryptophanyl-tRNA synthetase Met 1 –Gln 471 1.8 pNIC-CTB10H 153058 YARS TyrRS HA 8565 Tyrosyl-tRNA synthetase Met 1 –Ser 528 24.9 pNIC-CTB10H 153059 YARS2 TyrRS mt 51067 Tyrosyl-tRNA synthetase 2 mitochondrial Ala 31 –Leu 477 2.6 pNIC-CTB10H 153060 Open in a separate window List of aminoacyl-tRNA synthetases with antigen details Clinical names are used for descriptions of aaRS as autoantigens in idiopathic inflammatory myopathies.

Techniques: Purification, Expressing, Construct, Plasmid Preparation