Rate of helix formation by intracellular procollagen and protocollagen. Evidence for a role for disulfide bonds

Matrix-free cells from embryonic tendons were incubated under conditions in which they synthesized and accumulated protocollagen, the unhydroxylated form of procollagen, which is non-helical at 37°. Limited digestion with pepsin demonstrated that when the accumulated protocollagen was hydroxylated intracellularly to procollagen, or when the cells were cooled below the T_m of protocollagen, the protein became triple-helical in about 5 min, or in a fraction of the time required for isolated α chains to become helical. When disulfide bonds in the NH₂-terminal extensions of protocollagen were reduced by treating the cells with dithiothreitol, the rate of helix formation was markedly decreased. The results demonstrated that the NH₂-terminal extensions found in protocollagen and procollagen play an important role in formation of the triple-helix during biosynthesis.     

The effect on Raman spectra of extraction of peripheral proteins from human erythrocyte membranes

We compare the Raman spectra of intact erythrocyte membranes with spectra of membranes from which essentially all peripheral proteins have been extracted. The results indicate that the extraction procedure causes considerable alteration in the environment of peptide bonds and a marked change in the environment of the phenylalanine and tryptophan ring moieties of the integral membrane proteins. We infer from our data that the lipid portion of the bilayer is little affected by extraction. Finally, we have observed that the total decrease in the heights of the protein-associated peaks of the extracted vesicles is generally less than that expected from the mass of protein removed by extraction.     

Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110

Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell–cell communications and host–microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs.     

Detection of the associated state of membrane proteins by polyacrylamide gradient gel electrophoresis with non-denaturing detergents Application to band 3 protein from erythrocyte membranes

Polyacrylamide gradient gel electrophoresis was carried out in micellar solutions of various detergents which differ in degree of potency to denature proteins. From the application of this method to band 3 protein from erythrocyte membranes, it was suggested that the procedure was useful in studying the molecular state of membrane proteins.The electrophoretic behaviors of human and bovine band 3 protein did not show any species specificity in either a denature state and a state resembling the native state. As well as in nonionic detergent solutions, the dimeric and tetrameric structures of bovine band 3 protein were preserved in sodium deoxycholate solution, in which protein complexes maintained in nonionic detergent solutions are frequently dissociated. Even in dodecyltrimethylammonium bromide solution, which is a denaturant for water-soluble proteins, part of the band 3 protein was still present as the oligomer. The results suggest that the oligomeric form of band 3 protein is the stable structure and that the dimer and tetramer possibly coexist in membranes.     

A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.     

Purification and molecular properties of 3 polypeptides released from a highly active O2-evolving photosystem-II preparation by Tris-treatment

Tris-treatment of a highly active O₂-evolving photosystem-II preparation induced release of 3 polypeptides (M_r 33 000, 24 000 and 18 000), concomitant with inhibition of O₂ evolution [FEBS Lett. (1981)_133. 265-268]. The 3 polypeptides were purified with the use of electrofocusing. Isoelectric points of the proteins were 5.1, 6.5 and 9.2 in order of decreasing M_r value. Only a trace amount of histidine, cystein and methionine were detected in these proteins. Based on the amino acid compositions, polarity indexes of the proteins were calculated to be 47–49%, suggesting the 3 proteins to be hydrophilic.     

Improved method for cloning DNA complementary to minor mRNAs: preparation of a hybridization probe from purified mRNAs encoding intermediate filament proteins

A procedure for the rapid fractionation of mRNA has been used to enrich mRNAs encoding a set of intermediate filament proteins in trophoblastoma cells. The procedure involves sucrose-gradient fractionation followed by high-resolution preparative gel electrophoresis. Part of the enriched mRNA preparation has been used to prepare a hybridization probe to screen a trophoblastoma cDNA library in Escherichia coli. A small proportion of the clones hybridized to the probe, and among these a specific clone was identified.     

Disruption of alpha-mannosidase processing induces non-canonical hybrid-type glycosylation

Golgi alpha-mannosidase II is essential for the efficient formation of complex-type glycosylation. Here, we demonstrate that the disruption of Golgi alpha-mannosidase II activity by swainsonine in human embryonic kidney cells is capable of inducing a novel class of hybrid-type glycosylation containing a partially processed mannose moiety. The discovery of 'Man(6)-based' hybrid-type glycans reveals a broader in vivo specificity of N-acetylglucosaminyltransferase I, further defines the arm-specific tolerance of core alpha1-6 fucosyltransferase to terminal alpha1-2 mannose residues, and suggests that disruption of Golgi alpha-mannosidase II activity is capable of inducing potentially 'non-self' structures.     

Detailed N-glycan analysis of mannose receptor purified from murine spleen indicates tissue specific sialylation

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognises both mannosylated and sulphated ligands through its C-type lectin domains (CTLDs) and cysteine-rich (CR) domain, respectively. It is widely expressed among different tissues and by certain cell types in vivo. Our previous study suggested that the glycosylation, especially terminal sialylation, regulated the functional specificities of MR. In the current investigation, the distribution of MR among various mouse tissues was studied and the N-linked glycosylation of spleen MR was analysed. Our results showed that spleen expressed the most abundant MR, consistent with its wide distribution in different cell types in this organ. Spleen MR was heterogeneously N-glycosylated. The majority of the glycans were sialylated in the α2 → 6-linkage and both Neu5Ac and Neu5Gc sialic acids were detected. Most glycans were bi-antennary (74%) with ∼22% tri-antennary and most were core fucosylated (68%). About 13% contained α-galactose. In the lung, MR exhibited more terminal sialic acids in the α2 → 3- rather than in the α2 → 6-configuration. Our study provides a profile of MR N-linked glycosylation that will facilitate our understanding of their physiological role on MR biology in vivo.     

Glycoproteomics: Past, present and future

This invited paper reviews the study of protein glycosylation, commonly known as glycoproteomics, beginning with the origins of the subject area in the early 1970s shortly after mass spectrometry was first applied to protein sequencing. We go on to describe current analytical approaches to glycoproteomic analyses, with exemplar projects presented in the form of the complex story of human glycodelin and the characterisation of blood group H eptitopes on the O-glycans of gp273 from Unio elongatulus. Finally, we present an update on the latest progress in the field of automated and semi-automated interpretation and annotation of these data in the form of GlycoWorkBench, a powerful informatics tool that provides valuable assistance in unravelling the complexities of glycoproteomic studies.