Proteomic analysis of mammalian oligosaccharyltransferase reveals multiple subcomplexes that contain Sec61, TRAP, and two potential new subunits

Oligosaccharyltransferase (OST) catalyzes the cotranslational transfer of high-mannose sugars to nascent polypeptides during N-linked glycosylation in the rough endoplasmic reticulum lumen. Nine OST subunits have been identified in yeast. However, the composition and organization of mammalian OST remain unclear. Using two-dimensional Blue Native polyacrylamide gel electrophoresis/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry, we now demonstrate that mammalian OST can be isolated from solubilized, actively engaged ribosomes as multiple distinct protein complexes that range in size from approximately 500 to 700 kDa. These complexes exhibit different ribosome affinities and subunit compositions. The major complex, OSTC(I), had an apparent size of approximately 500 kDa and was readily released from ribosome translocon complexes after puromycin treatment under physiological salt conditions. Two additional complexes were released only after treatment with high salt: OSTC(II) ( approximately 600 kDa) and OSTC(III) ( approximately 700 kDa). Both remained stably associated with heterotrimeric Sec61alphabetagamma, while OSTC(III) also contained the tetrameric TRAP complex. All known mammalian OST subunits (STT3-A, ribophorin I, ribophorin II, OST48, and DAD1) were present in all complexes. In addition, two previously uncharacterized proteins were also copurified with OST. Mass spectrometry identified a 17 kDa protein as DC2 which is weakly homologous to the C-terminal half of yeast Ost3p and Ost6p. The second protein (14 kDa) was tentatively identified as keratinocyte-associated protein 2 (KCP2) and has no previously known function. Our results identify two potential new subunits of mammalian OST and demonstrate a remarkable heterogeneity in OST composition that may reflect a means for controlling nascent chain glycosylation.     

Prevascularization of collagen-glycosaminoglycan scaffolds: stromal vascular fraction versus adipose tissue-derived microvascular fragments

Background

The seeding of scaffolds with the stromal vascular fraction (SVF) of adipose tissue is a common prevascularization strategy in tissue engineering. Alternatively, adipose tissue-derived microvascular fragments (ad-MVF) may serve as vascularization units. In contrast to SVF single cells, they represent a mixture of intact arteriolar, capillary and venular vessel segments. Therefore, we herein hypothesized that the ad-MVF-based prevascularization of scaffolds is superior to the conventional SVF single cells-based approach.

Results

SVF single cells and ad-MVF were enzymatically isolated from epididymal fat pads of green fluorescent protein (GFP)⁺ donor mice to assess their viability and cellular composition using fluorescence microscopy and flow cytometry. Moreover, collagen-glycosaminoglycan matrices (Integra®) were seeded with identical amounts of the isolates and implanted into full-thickness skin defects within dorsal skinfold chambers of GFP^? recipient mice for the intravital fluorescent microscopic, histological and immunohistochemical analysis of implant vascularization and incorporation throughout an observation period of 2 weeks. Non-seeded matrices served as controls. While both isolates contained a comparable fraction of endothelial cells, perivascular cells, adipocytes and stem cells, ad-MVF exhibited a significantly higher viability. After in vivo implantation, the vascularization of ad-MVF-seeded scaffolds was improved when compared to SVF-seeded ones, as indicated by a significantly higher functional microvessel density. This was associated with an enhanced cellular infiltration, collagen content and density of CD31⁺/GFP⁺ microvessels particularly in the center of the implants, demonstrating a better incorporation into the surrounding host tissue. In contrast, non-seeded matrices exhibited a poor vascularization, incorporation and epithelialization over time.

Conclusions

The present study demonstrates that ad-MVF are highly potent vascularization units that markedly accelerate and improve scaffold vascularization when compared to the SVF.

     

Effects of redox potential and Ca2+ on the inositol 1,4,5-trisphosphate receptor L3-1 loop region: implications for receptor regulation

Inositol 1,4,5-trisphosphate receptor (IP(3)R) is a major intracellular Ca(2+) channel, modulated by many factors in the cytosolic and lumenal compartments. Compared with cytosolic control, lumenal-side regulation has been much less studied, and some of its mechanistic aspects have been controversial. Of particular interest with regard to lumenal regulation are whether it involves direct interactions between IP(3)R and the regulators, and whether it involves conformational changes of the lumenal regions of IP(3)R. To understand these lumenal-side regulation mechanisms, we studied the effects of two important lumenal regulatory factors, the redox potential and Ca(2+), on the L3-1 lumenal loop region of IP(3)R. The redox potential exerted direct and significant effects on the conformation of the loop region. By sharp contrast, Ca(2+) showed little effect on the L3-1 conformation, suggesting that the regulation of Ca(2+) is indirect or involves other receptor regions. GSH/oxidized glutathione-mediated oxidation introduced a unique intramolecular disulfide bond between Cys(34) and Cys(42). A variety of NMR experiments revealed that oxidation also induces localized helical characteristics in the Cys(34)-Cys(42) region. Dynamics studies also showed reduced motions in the region upon oxidation, consistent with the conformational changes. The results raise the interesting possibility that Cys(34) and Cys(42) may act together as a reduction sensor, and that Cys(65) may function as an oxidation sensor. Overall, our studies suggest that the redox potential and Ca(2+) can regulate IP(3)R through totally different mechanisms: Ca(2+) by the indirect effect and the redox potential by direct action causing conformational changes.     

Cross-platform comparability of microarray technology: Intra-platform consistency and appropriate data analysis procedures are essential

Background

The acceptance of microarray technology in regulatory decision-making is being challenged by the existence of various platforms and data analysis methods. A recent report (E. Marshall, Science, 306, 630–631, 2004), by extensively citing the study of Tan et al. (Nucleic Acids Res., 31, 5676–5684, 2003), portrays a disturbingly negative picture of the cross-platform comparability, and, hence, the reliability of microarray technology.

Results

We reanalyzed Tan's dataset and found that the intra-platform consistency was low, indicating a problem in experimental procedures from which the dataset was generated. Furthermore, by using three gene selection methods (i.e., p-value ranking, fold-change ranking, and Significance Analysis of Microarrays (SAM)) on the same dataset we found that p-value ranking (the method emphasized by Tan et al.) results in much lower cross-platform concordance compared to fold-change ranking or SAM. Therefore, the low cross-platform concordance reported in Tan's study appears to be mainly due to a combination of low intra-platform consistency and a poor choice of data analysis procedures, instead of inherent technical differences among different platforms, as suggested by Tan et al. and Marshall.

Conclusion

Our results illustrate the importance of establishing calibrated RNA samples and reference datasets to objectively assess the performance of different microarray platforms and the proficiency of individual laboratories as well as the merits of various data analysis procedures. Thus, we are progressively coordinating the MAQC project, a community-wide effort for microarray quality control.

     

CACA-TOCSY with alternate <Superscript>13</Superscript>C–<Superscript>12</Superscript>C labeling: a <Superscript>13</Superscript>C<Superscript>α</Superscript> direct detection experiment for mainchain resonance assignment,dihedral angle information,and amino acid type identification

We present a ¹³C direct detection CACA-TOCSY experiment for samples with alternate ¹³C–¹²C labeling. It provides inter-residue correlations between ¹³C^α resonances of residue i and adjacent C^αs at positions i − 1 and i + 1. Furthermore, longer mixing times yield correlations to C^α nuclei separated by more than one residue. The experiment also provides C^α-to-sidechain correlations, some amino acid type identifications and estimates for ψ dihedral angles. The power of the experiment derives from the alternate ¹³C–¹²C labeling with [1,3-¹³C] glycerol or [2-¹³C] glycerol, which allows utilizing the small scalar ³J_CC couplings that are masked by strong ¹J_CC couplings in uniformly ¹³C labeled samples.     

A case of spontaneous coronary artery disease regression

Coronary angiographic trials have demonstrated that lowering cholesterol can slow the progression of atherosclerosis, limit the formation of new lesions and enhance atherosclerotic regression together with reducing the incidence of clinical events (Waters D, 1996). Spontaneous regression of coronary atherosclerotic lesions is rare. We report the case of a patient with a severe within-stent restenotic lesion whose coronary disease spontaneously regressed 12 months after initial diagnosis, allowing for medical treatment of symptoms rather than repeated intervention. (Int J Cardiovasc Interventions 1999; 2: 121-123)     

Seq4SNPs: new software for retrieval of multiple, accurately annotated DNA sequences, ready formatted for SNP assay design

Background  

In moderate-throughput SNP genotyping there was a gap in the workflow, between choosing a set of SNPs and submitting their sequences to proprietary assay design software, which was not met by existing software. Retrieval and formatting of sequences flanking each SNP, prior to assay design, becomes rate-limiting for more than about ten SNPs, especially if annotated for repetitive regions and adjacent variations. We routinely process up to 50 SNPs at once.     

Mapping of the auto-inhibitory interactions of protein kinase R by nuclear magnetic resonance

The dsRNA-dependent protein kinase (PKR) is a key mediator of the anti-viral and anti-proliferative effects of interferon. Unphosphorylated PKR is characterized by inhibitory interactions between the kinase and RNA binding domains (RBDs), but the structural details of the latent state and its unraveling during activation are not well understood. To study PKR regulation by NMR we assigned a large portion of the backbone resonances of the catalytically inactive K296R kinase domain, and performed (15)N-heteronuclear single quantum coherence (HSQC) titrations of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2alpha docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2alpha docking site, F495A, displays weaker interactions with the RBD. The full-length RBD1+2 binds more strongly to the kinase domain than RBD2 alone. The observed chemical shift changes extend from the eIF2alpha binding site into the kinase N-lobe and inside the active site, consistent with weak interactions between the N-terminal part of the RBD and the kinase.     

Estimation of divergence time between two sibling species of the <Emphasis Type="Italic">Anopheles (Kerteszia) cruzii</Emphasis>complex using a multilocus approach

Background  

Anopheles cruzii is the primary human Plasmodium vector in southern and southeastern Brazil. The distribution of this mosquito follows the coast of the Brazilian Atlantic Forest. Previous studies indicated that An. cruzii is a complex of cryptic species.