improved-gel-reductive-elimination-for-comprehensive-o-linked-sulfo

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

University of Georgia

The beta-1,4-galactosyltransferase (beta-1,4-GalT) V whose human and mouse genes were cloned by us has been suggested to be involved in the biosynthesis of N-glycans and O-glycans, and lactosylceramide. To determine its biological function, beta-1,4-GalT V (B4galt5) mutant mice obtained by a gene trap method were analyzed. Analysis of pre- and post-implantation embryos revealed that the B4galt5(-/-) mice die by E10.5 while B4galt5(+/-) mice were born and grown normally. Histological study showed that most tissues are formed in B4galt5(-/-) embryos but their appearance at E10.5 is close to that of B4galt5(+/-) embryos at E9.0-9.5. The results indicate that the growth is delayed by one to one and half day in B4galt5(-/-) embryos when compared to B4galt5(+/-) embryos, which results in early death of the embryos by E10.5, probably due to hematopoietic and/or placental defects.

Early lethality of beta-1,4-galactosyltransferase V-mutant mice by growth retardation.

Human Î²-1,4-galactosyltransferase (Î²-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from Î²-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the Î²-1,4-GalT I-VI genes revealed that the expression of the Î²-1,4-GalT V gene in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells are a half and null when compared to that of B4galt5 ( +/+ )-derived MEF cells without altering the expression levels of other Î²-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When Î²-1,4-GalT activities were determined towards GlcNAcÎ²-S-pNP, no significant difference in its specific activity was obtained among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[(3)H]Gal, Lac-Cer synthase activity in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells decreased to 41% and 11% of that of B4galt5 ( +/+ )-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5 ( -/- ) -derived MEF cells decreased remarkably when compared with those of B4galt5 ( +/+ )-derived MEF cells. These results indicate that murine Î²-1,4-GalT V is involved in Lac-Cer biosynthesis.

Involvement of murine Î²-1,4-galactosyltransferase V in lactosylceramide biosynthesis.

Humaine &beta;-1, 4-galactosyltransf&eacute;rase (&beta;-1, 4-GalT) V s&#39;est av&eacute;r&eacute; &ecirc;tre impliqu&eacute;s dans la biosynth&egrave;se des N-glycans, O-glycanes et lactosylc&eacute;ramide (Lac-Cer) par des &eacute;tudes in vitro. Pour d&eacute;terminer sa sp&eacute;cificit&eacute; de substrat, l&#39;activit&eacute; enzymatique et ses produits ont &eacute;t&eacute; analys&eacute;s &agrave; l&#39;aide de cellules de fibroblastes embryonnaires (MEF) de souris de &beta;-1, 4-GalT V (B4galt5)-souris mutantes. Analyse des niveaux d&#39;expression des g&egrave;nes &beta;-1, 4-GalT I-VI a r&eacute;v&eacute;l&eacute; que l&#39;expression du g&egrave;ne de la &beta;-1, 4-GalT V dans B4galt5 (+/-) - et B4galt5 (- / -) - d&eacute;riv&eacute;es des cellules de force exp&eacute;ditionnaire des marines sont demi et null lorsque compar&eacute;e &agrave; celle de B4galt5 (+ / +)-d&eacute;riv&eacute; des cellules de force exp&eacute;ditionnaire des marines sans modifier les niveaux d&#39;expression d&#39;autres g&egrave;nes &beta;-1, 4-GalT. Ces cellules de force exp&eacute;ditionnaire des marines ne montr&eacute;s aucune diff&eacute;rence apparente dans leur croissance. Lorsque les activit&eacute;s de &beta;-1, 4-GalT &eacute;taient d&eacute;termin&eacute;es vers GlcNAc&beta;-S-pNP, aucune diff&eacute;rence significative de son activit&eacute; sp&eacute;cifique a &eacute;t&eacute; obtenue chez les B4galt5 (+ / +)-, B4galt5 (+/-) - et B4galt5 (- / -) - d&eacute;riv&eacute;s des cellules de force exp&eacute;ditionnaire des marines. Pas de diff&eacute;rences significatives ont &eacute;t&eacute; obtenues dans les structures et les quantit&eacute;s de N-glycanes et lectine liaisons aux glycoprot&eacute;ines membranaires chez B4galt5 (+ / +)-, B4galt5 (+/-) - et B4galt5 (- / -) - d&eacute;riv&eacute;s des cellules de force exp&eacute;ditionnaire des marines. Cependant, lorsque les homog&eacute;nats cellulaires sont incub&eacute;s avec glucosylc&eacute;ramide en pr&eacute;sence de UDP-[3 H] Gal, Lac-Cer l&#39;activit&eacute; synthase dans B4galt5 (+/-) - et B4galt5 (- / -) - d&eacute;riv&eacute;s des cellules de force exp&eacute;ditionnaire des marines a chut&eacute; &agrave; 41 % et 11 % de celle de B4galt5 (+ / +)-d&eacute;riv&eacute; des cellules de force exp&eacute;ditionnaire des marines. Conforme ce, montants de Lac-Cer et ses d&eacute;riv&eacute;s GM3 en B4galt5 (- / -) - d&eacute;riv&eacute;es des cellules de force exp&eacute;ditionnaire des marines diminue remarquablement comparativement &agrave; celles de B4galt5 (+ / +)-d&eacute;riv&eacute; des cellules de force exp&eacute;ditionnaire des marines. Ces r&eacute;sultats indiquent que murin V &beta;-1, 4-GalT est impliqu&eacute;e dans la biosynth&egrave;se de Lac-Cer.

Participation de murin V &beta;-1, 4-galactosyltransf&eacute;rase lactosylc&eacute;ramide biosynth&egrave;se.

&Beta;-1,4-galattosiltransferasi umana (&beta;-1,4-GalT) V ha dimostrato di essere coinvolti nella biosintesi di N-glicani, O-glicani e lactosylceramide (Lac-Cer) da studi in vitro. Per determinare la specificit&agrave; di substrato, l&#39;attivit&agrave; enzimatica e i suoi prodotti sono stati analizzati utilizzando cellule embrionali del fibroblasto (MEF) del mouse da GalT-&beta;-1,4 V (B4galt5)-topi mutanti. Analisi dei livelli di espressione dei geni &beta;-1,4-GalT VI ha rivelato che l&#39;espressione del gene &beta;-1,4-GalT V in B4galt5 (+ /-) - e B4galt5 (- / -) - derivate le cellule MEF sono un mezzo e null quando paragonato a quello di B4galt5 (+ +)-derivato celle MEF senza alterare i livelli di espressione di altri geni &beta;-1,4-GalT. Queste cellule MEF ha mostrato alcuna differenza apparente nella loro crescita. Quando verso GlcNAc&beta;-S-pNP sono state determinate attivit&agrave; &beta;-1,4-GalT, nessuna differenza significativa nella sua attivit&agrave; specifica &egrave; stata ottenuta tra B4galt5 (+ +)-, B4galt5 (+ /-) - e B4galt5 (- / -) - cellule del MEF. Differenze significative sono state ottenute in strutture e quantit&agrave; di N-glicani e lectina associazioni di glicoproteine di membrana tra B4galt5 (+ +)-, B4galt5 (+ /-) - e B4galt5 (- / -) - cellule del MEF. Tuttavia, quando la cella omogeneati sono stati incubati con glucosilceramidi in presenza di B4galt5 e UDP-[(3) H] Gal, Lac-Cer sintasi attivit&agrave; in B4galt5 (+ /-)-(- / -) - cellule MEF scesa al 41% e l&#39;11% di quello di B4galt5 (+ +)-cellule MEF. Coerente con questa, gli importi di Lac-Cer e suoi derivati GM3 in B4galt5 (- / -) - derivate le celle del MEF &egrave; diminuito notevolmente quando confrontati con quelli di B4galt5 (+ +)-cellule MEF. Questi risultati indicano che V &beta;-1,4-GalT murino &egrave; coinvolto nella biosintesi di Lac-Cer.

Coinvolgimento di murino &beta;-1,4-galattosiltransferasi V nella biosintesi di lactosylceramide.

Humana &beta;-1, 4-galactosyltransferase (&beta;-1, 4-GalT) V fue demostrado para ser implicados en la bios&iacute;ntesis de N-glicanos, O-glicanos y lactosylceramide (Lac-Cer) por estudios in vitro. Para determinar su especificidad de sustrato, actividad enzim&aacute;tica y sus productos fueron analizados utilizando c&eacute;lulas de fibroblastos embrionarios (MEF) de rat&oacute;n de &beta;-1, 4-GalT V (B4galt5)-ratones mutantes. El an&aacute;lisis de los niveles de expresi&oacute;n de los genes de la &beta;-1, 4-GalT VI revel&oacute; que la expresi&oacute;n del gen &beta;-1, 4-GalT V en B4galt5 (+/-) y B4galt5 (- / -) - c&eacute;lulas derivadas de MEF son medio y nulo en comparaci&oacute;n con la de B4galt5 (+ / +)-derivado de las c&eacute;lulas MEF sin alterar los niveles de expresi&oacute;n de otros genes de &beta;-1, 4-GalT. Estas c&eacute;lulas MEF no mostraron ninguna diferencia aparente en su crecimiento. Cuando se determinaron las actividades de la &beta;-1, 4-GalT hacia GlcNAc&beta;-S-pNP, se obtuvieron diferencias significativas en su actividad espec&iacute;fica entre B4galt5 (+ / +)-, B4galt5 (+/-) y B4galt5 (- / -) - derivado de las c&eacute;lulas MEF. No hubo diferencias significativas se obtuvieron en las estructuras y las cantidades de N-glicanos y lectinas enlaces a glicoprote&iacute;nas de la membrana entre B4galt5 (+ / +)-, B4galt5 (+/-) y B4galt5 (- / -) - derivado de las c&eacute;lulas MEF. Sin embargo, cuando los homogenados celulares se incubaron con glucosilceramida en presencia de UDP-[3 H] Gal, Lac-Cer synthase actividad en B4galt5 (+/-) y B4galt5 (- / -) - derivado de las c&eacute;lulas MEF se redujo a 41% y 11% de la de B4galt5 (+ / +)-derivado de las c&eacute;lulas MEF. Consistente con este, cantidades de Lac-Cer y sus derivados GM3 en B4galt5 (- / -) - derivado c&eacute;lulas MEF disminuyeron notablemente en comparaci&oacute;n con los de B4galt5 (+ / +)-derivado de las c&eacute;lulas MEF. Estos resultados indican que murino &beta;-1, 4-GalT V participa en la bios&iacute;ntesis de Lac-Cer.

Participaci&oacute;n de murino V de &beta;-1, 4-galactosyltransferase en la bios&iacute;ntesis de lactosylceramide.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique for protein separation, and in-gel tryptic digestion of resolved protein bands has enhanced the resolution of protoeomic analysis. To augment this technology and expand its usefulness for glycoproteomics, we have developed and improved methods to release and recover O-linked glycans from proteins resolved in SDS-PAGE gels for subsequent analysis by mass spectrometry (MS). Gel pieces containing target proteins are washed to remove contaminants. O-linked glycans are released through reductive β-elimination by hydrating gel pieces in base and adding reductant. Following straightforward sample cleanup, this simple treatment of glycoprotein gel pieces produces material suitable for MS analysis. We have applied this method to the analysis of mucin-type glycoproteins that are expected to carry high densities of sialylated and sulfated O-linked glycans. However, the strongly acidic nature of the sulfate moiety suppresses MS signal intensities, hampering detection and quantitative analysis. To enhance detection, we present an improved method for sulfoglycomics. A mixture of sulflo-, sialo-, and neutral glycans were permethylated and partitioned into a water-dichloromethane (DCM) solvent mixture. Sulfated glycans were selectively recovered from the aqueous phase, while neutral and sialylated glycans remained in the DCM phase. When applied to the analysis of human mucin salivary glycans, this partition method generated material of sufficient quality to identify more than 60 glycan structures by NSI-MS (LTQ-Orbitrap) in positive and negative ion modes. Also, nearly 100% of the sulfated O-linked glycans were recovered in the aqueous phase, demonstrating the feasibility of in-depth sulfoglycomic analysis using SDS-PAGE resolved proteins.

Tadahiro Kumagai

Complex Carbohydrate Research Center

University of Georgia

Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Tadahiro Kumagai

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Complex Carbohydrate Research Center

University of Georgia

Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Complex Carbohydrate Research Center