Probing platelet factor 4 alpha-granule targeting.

The storage mechanism of endogenous secretory proteins in megakaryocyte alpha-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet alpha-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in alpha-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify alpha-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in alpha-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze alpha-granule biogenesis and its alterations in the congenital defect gray platelet syndrome.

cld and lec23 are disparate mutations that affect maturation of lipoprotein lipase in the endoplasmic reticulum.

The mutations cld (combined lipase deficiency) and lec23 disrupt in a similar manner the expression of lipoprotein lipase (LPL). Whereas cld affects an unknown gene, lec23 abolishes the activity of alpha-glucosidase I, an enzyme essential for proper folding and assembly of nascent glycoproteins. The hypothesis that cld, like lec23, affects the folding/assembly of nascent LPL was confirmed by showing that in cell lines homozygous for these mutations (Cld and Lec23, respectively), the majority of LPL was inactive, displayed heterogeneous aggregation, and had a decreased affinity for heparin. While inactive LPL was retained in the ER, a small amount of LPL that had attained a native conformation was transported through the Golgi and secreted. Thus, Cld and Lec23 cells recognized and retained the majority of LPL as misfolded, maintaining the standard of quality control. Examination of candidate factors affecting protein maturation, such as glucose addition and trimming, proteins involved in lectin chaperone cycling, and other abundant ER chaperones, revealed that calnexin levels were dramatically reduced in livers from cld/cld mice; this finding was also confirmed in Cld cells. We conclude that cld may affect components in the ER, such as calnexin, that play a role in protein maturation. Whether the reduced calnexin levels per se contribute to the LPL deficiency awaits confirmation.

Enhanced detection of lipoprotein lipase by combining immunoprecipitation with Western blot analysis.

This manuscript describes the problems inherent in combining immunoprecipitation of lipoprotein lipase (LPL) with its detection by Western blot, and how these problems can be circumvented by the preparation of suitable immunoreagents. These reagents used during the immunoprecipitation step, include Fab fragments of the primary antibody (chicken anti-bovine LPL), and a covalently linked immunomatrix of the secondary antibody (rabbit anti-chicken IgG). The use of these reagents in conjunction with Western blot detection virtually eliminates the problem of non-relevant protein detection when analyzing LPL from complex biological samples. Moreover, this approach can be adapted to detect any protein with the same inherent problems as LPL, such as hepatic lipase.

Evidence for a sustained genetic effect on fat storage capacity in cultured adipose cells from Zucker rats.

Using mature adipocytes and preadipocytes from genetically obese Zucker rats, we investigated the cells' ability to maintain abnormal fat storage capacity when withdrawn from their in vivo environment. Long-term adipocyte cultures from obese rats displayed an increase in both glucose consumption (GC) and enzyme activities, including fatty acid synthase (4-fold), glycerol-3-phosphate dehydrogenase (4.5-fold), lipoprotein lipase (LPL; 6-fold), and malic enzyme (2.5-fold). Fully differentiated obese predipocytes exhibited a twofold increase in these enzyme activities, together with higher glucose metabolism. In obese cells, LPL mRNA was increased in both adipocytes (6-fold) and differentiated preadipocytes (2-fold). Insulin mediated an increase in GC and lipogenic enzymes in both adipocytes and preadipocytes regardless of the genotype; this effect was more marked in obese cells. Examining cultured adipocytes from rats fed a high-fat diet, we showed that the nutritional effect upon GC and lipogenic enzymes was abolished after culture. These results demonstrated that fatty mutation may be intrinsically expressed in prolonged cultured mature adipocytes and in newly differentiated adipocytes.