protein bounds  (Clinx Science)

Journal: bioRxiv

Article Title: Discovery of dihydroxy-enone–type protein-bound ceramides as the dominant type in human stratum corneum

doi: 10.64898/2026.04.08.717327

Figure Lengend Snippet: Ceramide structures and proposed biosynthetic pathways for P-EO–type and P-O–type protein-bound ceramides. ( A ) Structures of non-acylated ceramides and acylceramides. ( B ) Proposed biosynthetic pathways for P-EO–type and P-O–type protein-bound ceramides. The linoleic acid moiety of acylceramides undergoes hydroperoxidation catalyzed by ALOX12B, followed by hydroperoxide isomerization by ALOXE3, resulting in the formation of EH acylceramides. EH acylceramides are then converted into EE acylceramides or TH acylceramides by SDR9C7 or epoxide hydrolases, including EPHX3, respectively. EE acylceramides covalently bind to cysteine residues of corneocyte surface proteins via Michael addition, yielding P-EO–type protein-bound ceramides (P-EO [EE]). In the proposed pathway for the formation of P-O–type protein-bound ceramides, the O -acyl chains of EH, EE, and/or TH acylceramides are cleaved by an as-yet-unidentified esterase to yield ω-hydroxyceramides, which are subsequently covalently bound to glutamate residues of corneocyte surface proteins. Although TGM1 has been considered as a candidate enzyme, its involvement in this process remains debated. Dashed arrows indicate putative pathways for which the responsible enzymes and/or reaction mechanisms remain unknown.

Article Snippet: The protein-bound ceramide fraction before oxidative sulfoxide elimination (total fraction), as well as the reversibly and irreversibly bound protein-bound ceramide fractions, were mixed with 400 μL of 1 M KOH in 95% CH 3 OH containing 1 pmol of N -(2’-( R )-hydroxypalmitoyl( d 9 ))-D- erythro -sphingosine ( d 9 -C16:0 α-hydroxyceramide; Avanti Polar Lipids) as an internal standard and incubated at 60 °C for 1 h to hydrolyze the ester linkages and release ω-hydroxyceramides from each fraction.

Techniques:

Journal: bioRxiv

Article Title: Discovery of dihydroxy-enone–type protein-bound ceramides as the dominant type in human stratum corneum

doi: 10.64898/2026.04.08.717327

Figure Lengend Snippet: Differential abundance of P-EO (DE) and P-EO (EE) ceramides in human SC and mouse epidermis. Protein-bound ceramide fractions were prepared from human SC ( A and B ) and P0 mouse epidermis ( C and D ). Following oxidative sulfoxide elimination, modified acylceramide moieties were released from P-EO–type ceramides and quantified via LC–MS/MS in the MRM mode. The total quantities of DE and EE acylceramides ( A and C ), quantities of the individual DE or EE acylceramide species categorized by N -acyl chain moiety ( B and D ), and the ratio of DE acylceramides relative to EE acylceramides ( E ) are shown. Values presented are means + SD (n = 4; ** P < 0.01; Welch’s t -test). n.d., not detected.

Article Snippet: The protein-bound ceramide fraction before oxidative sulfoxide elimination (total fraction), as well as the reversibly and irreversibly bound protein-bound ceramide fractions, were mixed with 400 μL of 1 M KOH in 95% CH 3 OH containing 1 pmol of N -(2’-( R )-hydroxypalmitoyl( d 9 ))-D- erythro -sphingosine ( d 9 -C16:0 α-hydroxyceramide; Avanti Polar Lipids) as an internal standard and incubated at 60 °C for 1 h to hydrolyze the ester linkages and release ω-hydroxyceramides from each fraction.

Techniques: Modification, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Discovery of dihydroxy-enone–type protein-bound ceramides as the dominant type in human stratum corneum

doi: 10.64898/2026.04.08.717327

Figure Lengend Snippet: Identification of DE-containing P-EO–type protein-bound ceramides in human SC. ( A – D ) Protein-bound ceramide fractions were prepared from human SC and P0 mouse epidermis, and modified acylceramide moieties were released from P-EO–type protein-bound ceramides by oxidative sulfoxide elimination, followed by LC–MS/MS analysis. Total ion chromatograms ( A ) and mass spectra ( B – D ), together with the corresponding ceramide structures and their characteristic ions ( A , C , and D ), are shown. ( A and B ) Precursor ion scanning analyses (Q3, m/z 264; scan range, m/z 900–1200; positive ion mode) were performed to detect ceramides with a sphingosine backbone, shown as total ion chromatograms of mouse epidermis and human SC ( A ) and the corresponding mass spectra for peaks 1 and 2 detected in the human SC chromatogram ( B ). ( C and D ) Product ion scanning analyses were performed in the negative ( C ; Q1, m/z 1,059 [human SC] and m/z 1,095 [mouse epidermis]; scan range, m/z 250–1,100) and positive ion modes ( D ; Q1, m/z 1,061 [human SC] and m/z 1,097 [mouse epidermis]; scan range, m/z 250–1,100). In the schematic diagrams, the long-chain base and N -acyl chain are shown in purple and blue, respectively. ( E ) Structures of protein-bound ceramides (P-EO [DE] ceramides) identified in human SC and of DE acylceramides released by oxidative sulfoxide elimination. The structures of P-EOS (DE) and EOS (DE) are shown; these represent the sphingosine-containing forms of P-EO [DE] ceramides and DE acylceramides, respectively. Ceramide classes are represented by combinations of abbreviations for the N -acyl chain and long-chain base moieties. EOS indicates ceramides composed of the N -acyl chain moiety EO ( e sterified ω -hydroxy fatty acid) and the long-chain base moiety S ( s phingosine).

Article Snippet: The protein-bound ceramide fraction before oxidative sulfoxide elimination (total fraction), as well as the reversibly and irreversibly bound protein-bound ceramide fractions, were mixed with 400 μL of 1 M KOH in 95% CH 3 OH containing 1 pmol of N -(2’-( R )-hydroxypalmitoyl( d 9 ))-D- erythro -sphingosine ( d 9 -C16:0 α-hydroxyceramide; Avanti Polar Lipids) as an internal standard and incubated at 60 °C for 1 h to hydrolyze the ester linkages and release ω-hydroxyceramides from each fraction.

Techniques: Modification, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Discovery of dihydroxy-enone–type protein-bound ceramides as the dominant type in human stratum corneum

doi: 10.64898/2026.04.08.717327

Figure Lengend Snippet: Age-dependent increases in Ephx3 expression and the (9 R ,10 S ) stereoisomer of P-EO (DE) ceramides in mouse epidermis. ( A ) Schematic representation of the conversion of the (9 R ,10 R ,13 R ) EH fatty acid to the (9 R ,10 S ,13 R ) TH fatty acid mediated by the epoxide hydrolase EPHX2 and EPHX3. ( B – D ) Total RNA ( B ) or protein-bound ceramide fractions ( C and D ) were prepared from the skin of mice at postnatal day 0 (P0), one month, and three months of age. ( B ) Expression levels of Ephx2 and Ephx3 were quantified via quantitative real-time RT–PCR and normalized to the housekeeping gene Hprt1 . Values presented are means + SD (n = 3; * P < 0.05, ** P < 0.01; Tukey’s test). ( C and D ) DE and EE acylceramides were released from the protein-bound ceramide fraction via oxidative sulfoxide elimination and individually quantified via LC–MS/MS in the MRM mode. ( C ) Representative MRM chromatogram of the DE acylceramide containing C34:1 N -acyl chain. Peaks A and B are presumed to correspond to the (9 R ,10 S ) and (9 R ,10 R ) stereoisomers, respectively. ( D ) Values presented are means + SD of the ratios of DE to EE acylceramide peak areas for peaks A and B, respectively (n = 3; * P < 0.05, ** P < 0.01; Tukey’s test). ( E ) Schematic illustration of a proposed stereospecific cleavage of EE acylceramides into DE acylceramides mediated by EPHX3. In the schematic diagrams, the long-chain base and N -acyl chain are shown in purple and blue, respectively.

Article Snippet: The protein-bound ceramide fraction before oxidative sulfoxide elimination (total fraction), as well as the reversibly and irreversibly bound protein-bound ceramide fractions, were mixed with 400 μL of 1 M KOH in 95% CH 3 OH containing 1 pmol of N -(2’-( R )-hydroxypalmitoyl( d 9 ))-D- erythro -sphingosine ( d 9 -C16:0 α-hydroxyceramide; Avanti Polar Lipids) as an internal standard and incubated at 60 °C for 1 h to hydrolyze the ester linkages and release ω-hydroxyceramides from each fraction.

Techniques: Expressing, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Discovery of dihydroxy-enone–type protein-bound ceramides as the dominant type in human stratum corneum

doi: 10.64898/2026.04.08.717327

Figure Lengend Snippet: Proposed biosynthetic pathways for P-EO–type protein-bound ceramides. Acylceramides are first converted into EH acylceramides through ALOX12B-dependent hydroperoxidation followed by ALOXE3-mediated hydroperoxide isomerization and are subsequently oxidized by SDR9C7 to yield EE acylceramides. A portion of EH acylceramides can be converted into TH acylceramides via epoxide opening catalyzed by EPHX family enzymes (e.g., EPHX3). Although this conversion is not predominant under physiological conditions, it can become dominant and lead to accumulation in patients with ichthyosis carrying SDR9C7 mutations ( , ). Predominantly in mice and partially in humans, EE acylceramides undergo Michael addition to cysteine residues of corneocyte surface proteins to form P-EO (EE) protein-bound ceramides. Alternatively, mainly in humans and partially in mice, EE acylceramides are converted into DE acylceramides by epoxide hydrolase(s) and subsequently form P-EO (DE) protein-bound ceramides via Michael addition. In addition, DE acylceramides may also be generated through a pathway in which EH acylceramides are first converted into TH acylceramides and then oxidized to DE acylceramides; both steps are shown as dashed arrows. In the schematic diagrams, the long-chain base and N -acyl chain are shown in purple and blue, respectively.

Article Snippet: The protein-bound ceramide fraction before oxidative sulfoxide elimination (total fraction), as well as the reversibly and irreversibly bound protein-bound ceramide fractions, were mixed with 400 μL of 1 M KOH in 95% CH 3 OH containing 1 pmol of N -(2’-( R )-hydroxypalmitoyl( d 9 ))-D- erythro -sphingosine ( d 9 -C16:0 α-hydroxyceramide; Avanti Polar Lipids) as an internal standard and incubated at 60 °C for 1 h to hydrolyze the ester linkages and release ω-hydroxyceramides from each fraction.

Techniques: Generated

Journal: bioRxiv

Article Title: Discovery of dihydroxy-enone–type protein-bound ceramides as the dominant type in human stratum corneum

doi: 10.64898/2026.04.08.717327

Figure Lengend Snippet: Abundance of reversibly and irreversibly bound protein-bound ceramides in human SC. ( A ) Schematic representation of both reversibly bound protein-bound ceramides that are released via oxidative sulfoxide elimination upon incubation of the protein-bound ceramide fraction in organic solvent and irreversibly bound protein-bound ceramides that remain unreleased. P-EO (EE) and P-EO (DE) ceramides are classified as reversibly bound protein-bound ceramides, whereas P-O ceramides are classified as irreversibly bound protein-bound ceramides. The irreversibly bound fraction may also include protein-bound ceramides derived from P-EO–type ceramides, although their structures have not yet been elucidated. ( B ) Schematic overview of the preparation and quantification of total, reversible, and irreversible fractions of protein-bound ceramides. The protein-bound ceramide fraction without treatment (total), the fraction released by oxidative sulfoxide elimination (reversible fraction), and the residual unreleased fraction (irreversible fraction) were each subjected to alkaline treatment to convert all types of protein-bound ceramides to ω-hydroxyceramides, which were then quantified via LC–MS/MS. ( C and D ) Protein-bound ceramide fractions were prepared from human SC, suspended in chloroform/methanol (1:2, v/v), and incubated at 60 °C to induce oxidative sulfoxide elimination. The incubation was performed six times (15 h for the third incubation and 3 h for the others). ( C ) Quantities of DE and EE acylceramides released in each eluate were quantified via LC–MS/MS in the MRM mode. Values presented are means + SD (n = 4). ( D ) The protein-bound ceramide fraction (total), the combined eluates from six incubations (reversible fraction), and the pellet remaining after the sixth incubation (irreversible fraction) were each subjected to alkaline treatment to convert all types of protein-bound ceramides to ω-hydroxyceramides, followed by quantification via LC–MS/MS in the MRM mode. Values presented are means + SD (n = 4).

Article Snippet: The protein-bound ceramide fraction before oxidative sulfoxide elimination (total fraction), as well as the reversibly and irreversibly bound protein-bound ceramide fractions, were mixed with 400 μL of 1 M KOH in 95% CH 3 OH containing 1 pmol of N -(2’-( R )-hydroxypalmitoyl( d 9 ))-D- erythro -sphingosine ( d 9 -C16:0 α-hydroxyceramide; Avanti Polar Lipids) as an internal standard and incubated at 60 °C for 1 h to hydrolyze the ester linkages and release ω-hydroxyceramides from each fraction.

Techniques: Incubation, Solvent, Derivative Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Discovery of dihydroxy-enone–type protein-bound ceramides as the dominant type in human stratum corneum

doi: 10.64898/2026.04.08.717327

Figure Lengend Snippet:

Article Snippet: The protein-bound ceramide fraction before oxidative sulfoxide elimination (total fraction), as well as the reversibly and irreversibly bound protein-bound ceramide fractions, were mixed with 400 μL of 1 M KOH in 95% CH 3 OH containing 1 pmol of N -(2’-( R )-hydroxypalmitoyl( d 9 ))-D- erythro -sphingosine ( d 9 -C16:0 α-hydroxyceramide; Avanti Polar Lipids) as an internal standard and incubated at 60 °C for 1 h to hydrolyze the ester linkages and release ω-hydroxyceramides from each fraction.

Techniques: