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H Ploegh

Publications and source records attributed to H Ploegh.

47 records · Page 3Linked to original sources
PubMed1987

Crosses of two independently derived transgenic mice demonstrate functional complementation of the genes encoding heavy (HLA-B27) and light (beta 2-microglobulin) chains of HLA class I antigens.

In man a number of diseases are associated with certain alleles of MHC antigens. The most pronounced example is ankylosing spondylitis, which is strongly associated with HLA-B27. As a first step towards a model system to study the basis of this association, transgenic mice were generated that showed cell surface expression of the HLA-B27 antigen biochemically indistinguishable from HLA-B27 antigen expressed on human cells. This result was obtained by crossing two independently derived strains of mice, one of which is transgenic for the HLA-B27 heavy chain gene, and the other carrying and expressing the human beta 2m gene. Examination of HLA-B27 and human beta 2m mRNA in various tissues shows the two genes to be expressed in a coordinate fashion. The mRNA levels follow those of endogenous H-2 Class I genes.

Animals↗
PubMed1986

Inhibition of N-linked oligosaccharide trimming mannosidases blocks human B cell development.

Deoxymannojirimycin (dMM) or swainsonine (SW), which block conversion of high-mannose to complex-type N-linked glycans, strongly inhibited the production of immunoglobulin (Ig) when added to cultures of human lymphocytes together with the polyclonal B cell activators pokeweed mitogen (PWM) and Staphylococcus aureus (SAC). To obtain the inhibitory effect, inhibitor had to be present during the first 36 h of culture. Addition at later timepoints was less effective and showed that neither inhibitor interfered with rate of production or secretion of Ig as such. Viability and proliferation of the lymphocytes, as defined by cell number and rate of DNA synthesis, were not influenced by the presence of dMM or SW, and no changes in the relative number of helper (T4+) or suppressor (T8+) cells were observed. Thus, for normal differentiation of human B lymphocytes into Ig secreting (plasma) cells in response to PWM and SAC, conversion of high-mannose to complex N-linked glycans is essential.

1-Deoxynojirimycin↗
PubMed1986

Determination of attachment sites for N-linked carbohydrate groups of class II histocompatibility alpha-chain and analysis of possible O-linked glycosylation of alpha- and gamma-chains.

Two glycopeptide fractions were isolated from a tryptic digest of the human class II antigen alpha-subunit, by chromatography on Lens culinaris and Ricinus communis lectin columns, respectively. Partial NH2-terminal sequence analysis of radiochemically labelled glycopeptide fractions allowed alignment with two stretches of the deduced DR alpha sequence, each encompassing a signal for N-linked glycosylation, i.e. Asn-X-Thr(Ser). The fraction displaying affinity for L. culinaris lectin was susceptible to the action of endo-beta-N-acetylglucosaminidase H whereas the fraction adsorbed to R. communis was not. Since DR alpha is known to carry only one N-linked carbohydrate chain in the high-mannose form, this glycan could thereby be mapped to Asn 78. Accordingly, the complex N-linked carbohydrate is attached to Asn 118. Moreover, analysis of material released from class II antigens and gamma-chains upon mild alkaline hydrolysis, indicates the presence of O-linked sugars on the alpha- and gamma- but not the beta-subunits.

Amino Acid Sequence↗
PubMed1986

Activation of human B cells: involvement of surface immunoglobulin as evidenced by two biochemically distinct types of response to Staphylococcus aureus.

If activation of human B cells by Staphylococcus aureus proceeds through interaction of surface immunoglobulin with Staphylococcal protein A, then immunoglobulins should be produced that are capable of binding to protein A as a consequence of such stimulation. In the present report it is shown that two biochemically distinct types of response to S. aureus are demonstrable in human peripheral blood lymphocytes. Two types of IgM are produced: IgM capable of binding to protein A, and IgM that does not bind and can be recovered by immunoprecipitation with anti-Ig antibodies. Cloned cell lines produce one of either type of Ig, but not both. Therefore, interaction of protein A with Ig alone cannot account for the stimulatory properties of S. aureus. When S. aureus is used in conjunction with pokeweek mitogen, a synergistic effect between the two mitogens is seen. Under conditions of optimal synergistic stimulation, the increase in immunoglobulin production is seen virtually exclusively in the category of molecules capable of binding to protein A. These results offer strong support for a model where optimal differentiation of human B cells to plasma cells is contingent upon receiving at least two signals: one signal is delivered to the surface immunoglobulin, and one signal delivered to the B cell in a T cell and/or monocyte dependent fashion (B cell growth and differentiation factors).

B-Lymphocytes↗
PubMed1985

A hydrophobic transmembrane segment at the carboxyl terminus of thy-1.

The mode of integration of the glycoprotein thy-1 within the cell membrane has been controversial due to an apparent lack of a transmembrane hydrophobic segment. Rat and mouse complementary DNA and genomic clones encoding the thy-1 molecule have been isolated and sequenced. These studies have enabled us to determine the intron-exon organization of the thy-1 gene. Furthermore, they have revealed the existence of a sequence which would encode an extra segment (31 amino acids) at the carboxyl terminus of the thy-1 molecule. These extra amino acids include a 20-amino acid hydrophobic segment which may be responsible for integration of thy-1 within the plasma membrane.

Amino Acid Sequence↗
PubMed1984

Inhibition of N-linked oligosaccharide trimming does not interfere with surface expression of certain integral membrane proteins.

The effects of 1-deoxynojirimycin (dNM) and 1-deoxymannojirimycin (dMM), inhibitors of oligosaccharide trimming glucosidase I and mannosidase I, respectively, on the biosynthesis of vesicular stomatitis virus G protein, influenza virus hemagglutinin, and human class I histocompatibility antigens were investigated. Although the oligosaccharides of these membrane glycoproteins were greatly altered, neither dNM nor dMM interferred with their surface expression, as determined by a variety of assays, including accessibility to proteases and antibodies; neither did these drugs inhibit production of infectious virus particles.

1-Deoxynojirimycin↗
PubMed1983

Effects of the glucosidase inhibitors nojirimycin and deoxynojirimycin on the biosynthesis of membrane and secretory glycoproteins.

The glucosidase inhibitors nojirimycin (NM) and 1-deoxynojirimycin (dNM) interfere with N-linked glycosylation. The effects of NM and dNM on the biosynthesis of secretory glycoproteins (IgD and IgM) and membrane glycoproteins (HLA-A, B, C and -DR antigens) have been examined. Whereas treatment of IgD- and IgM-producing cells with NM results in the transfer of drastically shortened oligosaccharide side chains, treatment with dNM inhibits trimming, most probably through interaction with glucosidase I and/or II. A comparison of NM and dNM with tunicamycin and the mannosidase inhibitor swainsonine (SW) show that each of the inhibitors interferes with N-linked glycosylation in a distinct manner. For both Ig and HLA antigens, the effects of SW are discernible at the final stages of glycan maturation only, whereas the effects of dNM are observed quite early in the biosynthetic process. The secretion of IgD, but not IgM, was blocked in dNM-treated cells. The HLA-A, B, C heavy chains synthesized by the Daudi cell line were degraded in an accelerated fashion in dNM-treated cells, but no effects were seen on the HLA-DR antigens in these cells. Although both SW and dNM interfere with trimming, further modifications of the oligosaccharide side chains occur, and show that the two processes are not obligately coupled. Glucosidase inhibitors such as NM and dNM, as well as the mannosidase inhibitor SW, allow modification of glycan structure, and may be used to study the biological role of glycoprotein oligosaccharides and their modifications.

1-Deoxynojirimycin↗
PubMedDate not supplied

Novel mannosidase inhibitor blocking conversion of high mannose to complex oligosaccharides.

Many secretory and membrane proteins are glycoproteins carrying asparagine-linked (N-linked) oligosaccharides. There are two types of N-linked glycans, referred to as high-mannose and complex type, respectively. Biosynthesis of N-linked glycans of the complex type proceeds via a high-mannose intermediate. After the initial transfer of a high-mannose oligosaccharide with the composition (Glc)3(Man)9(GlcNAc)2 from a lipid carrier to the nascent polypeptide chain, trimming reactions take place. Trimming glucosidases remove the glucose residues quantitatively and mannosidases IA/B and II can remove all but three mannose residues. After trimming, terminal sugars such as N-acetylglucosamine, galactose, sialic acid and fucose may be added and result in the conversion to a glycan of the complex type. Because suitable inhibitors were lacking, it was difficult to assess the importance of the trimming reactions for proper intracellular traffic, modification reactions other than the addition of terminal sugars, or as regulatory steps in glycoprotein processing. Here we describe the action of 1-deoxymannojirimycin (1,5-dideoxy-1,5-imino-D-mannitol, dMM; Fig. 1) on the biosynthesis of IgM and IgD. dMM is the mannose analogue of 1-deoxynojirimycin (dNM; Fig. 1), itself a glucosidase inhibitor. We present evidence that dMM is a mannosidase inhibitor. In vivo dMM inhibits the equivalent of the mannosidase IA/B activities and blocks conversion of high-mannose to complex oligosaccharides. It is the first such inhibitor to be reported. Interference with the biosynthetic pathway of N-linked glycans could prove to be a powerful way to manipulate carbohydrate structure in vivo.

1-Deoxynojirimycin↗