Investing glycosyltransferase mechanism definition

Methodologic ambiguities, however, have cast doubt on these findings. We demonstrate that Golgi elements with glycosyltransferases are packaged into vesicles and transported from the megakaryocyte cell body to the nascent platelets, where they distribute intracellularly and to the plasma membrane. In addition, we show that activation of platelets releases substantial glycosyltransferase activity and promotes the incorporation of exogenously added FITC-conjugated cytidine monophosphate-sialic acid FITC—CMP-SA into plasma proteins in vivo, demonstrating that platelets are a rich source of glycosylation enzymes.

Furthermore, our results indicate that platelets contain a sufficient pool of sugar nucleotides to support in vitro glycosylation reactions. Platelet preparation The preparation of human and mouse platelet-rich plasma was performed as described previously. All centrifuge steps included prostaglandin E1 to prevent platelet activation. Approval for blood drawing was obtained from the institutional review board of Brigham and Women's Hospital, and informed consent was obtained according to the Declaration of Helsinki.

Glycosyltransferase assays Polypeptide N-acetylgalactosaminyltransferase GalNAc-transferase or GalNAc-T and galactosyltransferase Gal-transferase or Gal-T assays, including sensitivity to lactalbumin, were performed as described previously. All experiments were repeated at least in triplicate. For details, please see supplemental Methods available on the Blood Web site; see the Supplemental Materials link at the top of the online article. An isotype-specific irrelevant Ab was used as control.

Experiments were repeated 4 times or more. To obtain murine megakaryocytes, livers were recovered from mouse fetuses at embryonic day 13, and single-cell suspensions were generated, immunostained, and analyzed as described previously.

Experiments were repeated at least 2 times. Assays were carried out using 0. For the identification of sialylated proteins and glycosylation status, glycosylated platelets were subjected to immunoblotting and lectin blotting as described in supplemental Methods. Platelets were analyzed by flow cytometry before and after activation to confirm shape change and P-selectin exposure. In selected experiments, Gal-T activity was used as a model to test the release of enzyme after activation with 3, 6, A separate set of experiments used secretions from platelets as the enzyme and donor sources.

Secretions were obtained from TRAP-activated platelets as described in the preceding paragraph. Altogether, our data suggest that: 1 glycosyltransferases can escape the Golgi and reach the PM; 2 at the PM, they undergo endocytosis to transit the early endosome, late endosome, and lysosome sequentially; and 3 most glycosyltransferases do not possess a post-Golgi retrieval pathway to return to the Golgi. A caveat of our study is that the amount of transfected glycosyltransferases is likely higher than endogenous expression.

As a commonly adopted approach, overexpression has been proven to be successful in the field of membrane trafficking despite the concern that it could saturate the transport machinery. However, as the trafficking of glycosyltransferases is still unclear, caution needs to be exercised when interpreting our results see Discussion for further details.

S2A , consistent with ED shedding. The culture medium and cell lysate were subjected to western blot analysis using an anti-GFP antibody. The culture medium and corresponding cell lysate were diluted to the same volume and subsequently subjected to immunoprecipitation using a rabbit anti-mCherry polyclonal antibody.

C Schematic showing the intracellular trafficking itinerary of ST. See the main text for details. The culture medium and corresponding cell lysate were diluted to the same volume and subsequently subjected to immunoprecipitation using a rabbit anti-GFP polyclonal antibody. The band intensity at each time point was normalized to the sum of the corresponding culture medium and cell lysate band intensities. Normalized band intensities from the culture medium red and the cell lysate black are plotted against CHX treatment time.

E,F The half-life of cellular ST is not substantially affected when lysosomal degradation is inhibited. Plots show quantification of the gel blots. Band intensities were measured and normalized to that at 0 h. We conducted the following experiment to investigate whether ED shedding can occur at the post-Golgi localization.

We found that the ED of surface-labeled ST was detected in the medium 0. Taking these data into account, we illustrate the proposed intracellular trafficking itinerary of ST in Fig. Note that we cannot rule out the possibility that cleavage takes place at the endolysosome, which subsequently undergoes exocytosis to release the ED Luzio et al. In the presence of cycloheximide CHX , which inhibits protein synthesis, we observed that the total cellular ST gradually disappeared with a half-life of 4.

We found that the half-life of ST-GFP remained roughly the same as the control in the presence of the lysosomal inhibitor bafilomycin A1 or chloroquine Fig. The Golgi residence time is a quantitative metric of the Golgi retention Without post-Golgi retrieval, the Golgi localization of most glycosyltransferases is primarily controlled by their retention at the Golgi. Similar to constitutive secretory cargo such as Tac and VSVG, the Golgi pool of a glycosyltransferase is determined by the net effect of the following three trafficking pathways, as we previously discussed Fig.

For a glycosyltransferase, because cycling between the ER and Golgi is relatively fast in comparison with Golgi export Rhee et al. When protein synthesis is inhibited by CHX treatment, the Golgi pool of glycosyltransferase, as monitored by fluorescence intensity within the Golgi region of interest ROI during live-cell imaging Fig.

We reasoned that Golgi retention should be inversely related to Golgi export — a strong Golgi export can be viewed as weak Golgi retention and vice versa. View large Download slide Golgi residence time is a metric for Golgi retention. A Schematic illustrating the trafficking pathways that contribute to the Golgi localization of a glycosyltransferase.

The Golgi pool of a glycosyltransferase is determined by the net effect of the biosynthetic input from the ER-to-Golgi pathway and depletion by the Golgi-to-ER and the Golgi-to-PM pathways. See main text for details.

The total intensity of the GFP signal within the Golgi ROI, It, was plotted against the time, t, and the Golgi residence time was calculated by fitting the plot with the first-order exponential decay function. See Materials and Methods for details.

GL2 shRNA is a non-targeting negative control. Error bars represent s. In each time series, the normalized initial or the first frame integrated intensity of the Golgi hereafter referred to as the initial intensity of the Golgi was used to denote the expression level of ST-GFP. We plotted the Golgi residence time against the initial intensity of the Golgi Fig. The Golgi residence time remains relatively constant within a small range of expression levels despite a decreasing trend.

Therefore, we consider the Golgi residence time as a metric characteristic of Golgi retention as a first approximation. To validate the usage of the Golgi residence time as a metric for Golgi retention, we measured the Golgi residence time of ST when its Golgi retention is compromised. The retromer complex, which functions in carrier biogenesis at the endosome during endosome-to-Golgi trafficking Lu and Hong, ; Mahajan et al.

A series of GFP-tagged swapping chimeras were hence generated Fig. Quantification was performed on the cells shown in C. E,F Golgi residence times of swapping chimeras. Western blots demonstrated that all chimeras had expected molecular weights when expressed in cells Fig.

Under fluorescence microscopy, they displayed different extents of Golgi and PM localization revealed by surface staining Fig. Therefore, all chimeras appeared to embed themselves in the membrane with their C termini exposed to the lumen or extracellular space, consistent with their type II transmembrane topology.

Their Golgi localization was quantified by the Golgi-to-cell intensity ratio Fig. Through live-cell imaging, the Golgi residence times of all swapping chimeras were determined and found to be less than that of ST Fig.

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