PubMed Health⌕ Search

Biomedical subjects

Raymond A Dwek

Publications and source records attributed to Raymond A Dwek.

At least 55 records · Page 3Linked to original sources
PubMed2005

Human serum IgM glycosylation: identification of glycoforms that can bind to mannan-binding lectin.

The glycoprotein IgM is the major antibody produced in the primary immune response to antigens, circulating in the serum both as a pentamer and a hexamer. Pentameric IgM has a single J chain, which is absent in the hexamer. The mu (heavy) chain of IgM has five N-linked glycosylation sites. Asn-171, Asn-332, and Asn-395 are occupied by complex glycans, whereas Asn-402 and Asn-563 are occupied by oligomannose glycans. The glycosylation of human polyclonal IgM from serum has been analyzed. IgM was found to contain 23.4% oligomannose glycans GlcNAc2Man5-9, consistent with 100% occupancy of Asn-402 and 17% occupancy of the variably occupied site at Asn-563. Mannan-binding lectin (MBL) is a member of the collectin family of proteins, which bind to oligomannose and GlcNAc-terminating structures. A commercial affinity chromatography resin containing immobilized MBL has been reported to be useful for partial purification of mouse and also human IgM. Human IgM glycoforms that bind to immobilized MBL were isolated; these accounted for only 20% of total serum IgM. Compared with total serum IgM, the MBL-binding glycoforms contained 97% more GlcNAc-terminating structures and 8% more oligomannose structures. A glycosylated model of pentameric IgM was constructed, and from this model, it became evident that IgM has two distinct faces, only one of which can bind to antigen, as the J chain projects from the non-antigen-binding face. Antigen-bound IgM does not bind to MBL, as the target glycans appear to become inaccessible once IgM has bound antigen. Antigen-bound IgM pentamers therefore do not activate complement via the lectin pathway, but MBL might have a role in the clearance of aggregated IgM.

Acetylglucosamine↗
PubMed2005

Imino sugar inhibitors for treating the lysosomal glycosphingolipidoses.

The inherited metabolic disorders of glycosphingolipid (GSL) metabolism are a relatively rare group of diseases that have diverse and often neurodegenerative phenotypes. Typically, a deficiency in catabolic enzyme activity leads to lysosomal storage of GSL substrates and in many diseases, several other glycoconjugates. A novel generic approach to treating these diseases has been termed substrate reduction therapy (SRT), and the discovery and development of N-alkylated imino sugars as effective and approved drugs is discussed. An understanding of the molecular mechanism for the inhibition of the key enzyme in GSL biosynthesis, ceramide glucosyltransferase (CGT) by N-alkylated imino sugars, has also lead to compound design for improvements to inhibitory potency, bioavailability, enzyme selectivity, and biological safety. Following a successful clinical evaluation of one compound, N-butyl-deoxynojirimycin [(NB-DNJ), miglustat, Zavesca], for treating type I Gaucher disease, issues regarding the significance of side effects and CNS access have been addressed as exposure of drug to patients has increased. An alternative experimental approach to treat specific glycosphingolipid (GSL) lysosomal storage diseases is to use imino sugars as molecular chaperons that assist protein folding and stability of mutant enzymes. The principles of chaperon-mediated therapy (CMT) are described, and the potential efficacy and preclinical status of imino sugars is compared with substrate reduction therapy (SRT). The increasing use of imino sugars for clinical evaluation of a group of storage diseases that are complex and often intractable disorders to treat has considerable benefit. This is particularly so given the ability of small molecules to be orally available, penetrate the central nervous system (CNS), and have well-characterized biological and pharmacological properties.

Animals↗
PubMed2005

Tyrosinase-related protein-2 and -1 are trafficked on distinct routes in B16 melanoma cells.

Tyrosinase related protein (TRP)-1 and -2 regulate the main steps in melanin synthesis and are immune targets in skin cancer or autoimmune pigmentary disorders. We found that ionophore monensin (Mon) and the quaternary amine chloroquine (CQ) discriminate between the traffic routes of TRP-2 and TRP-1. TRP-2 N-glycan processing is interrupted by Mon between ER and trans-Golgi, whereas this process continues for TRP-1. Mature TRP-2 is diverted by CQ treatment to a degradation pathway which depends on functional vacuolar ATPases. Conversely, the subcellular distribution and stability of TRP-1 were not affected by CQ. We propose that TRP-2 is sorted and trafficked in the early secretory pathway with a cargo which does not include TRP-1; post Golgi, TRP-2 intersects the endocytic pathway following a route via early endosomes, possibly by rapid recycling from the plasma membrane. These data show that highly structural homologous glycoproteins use distinct trafficking pathways in the same cell.

Animals↗
PubMed2005

Soluble tyrosinase is an endoplasmic reticulum (ER)-associated degradation substrate retained in the ER by calreticulin and BiP/GRP78 and not calnexin.

Tyrosinase is a type I membrane protein regulating the pigmentation process in humans. Mutations of the human tyrosinase gene cause the tyrosinase negative type I oculocutaneous albinism (OCAI). Some OCAI mutations were shown to delete the transmembrane domain or to affect its hydrophobic properties, resulting in soluble tyrosinase mutants that are retained in the endoplasmic reticulum (ER). To understand the specific mechanisms involved in the ER retention of soluble tyrosinase, we have constructed a tyrosinase mutant truncated at its C-terminal end and investigated its maturation process. The mutant is retained in the ER, and it is degraded through the proteasomal pathway. We determined that the mannose trimming is required for an efficient degradation process. Moreover, this soluble ER-associated degradation substrate is stopped at the ER quality control checkpoint with no requirements for an ER-Golgi recycling pathway. Co-immmunoprecipitation experiments showed that soluble tyrosinase interacts with calreticulin and BiP/GRP78 (and not calnexin) during its ER transit. Expression of soluble tyrosinase in calreticulin-deficient cells resulted in the export of soluble tyrosinase of the ER, indicating the calreticulin role in ER retention. Taken together, these data show that OCAI soluble tyrosinase is an ER-associated degradation substrate that, unlike other albino tyrosinases, associates with calreticulin and BiP/GRP78. The lack of specificity for calnexin interaction reveals a novel role for calreticulin in OCAI albinism.

Albinism, Oculocutaneous↗
PubMed2005

Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans.

Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular carcinoma (HCC). The diagnosis of HCC is usually made in the late stages of the disease, when treatment options are limited and prognosis is poor. We therefore have developed a method of glycoproteomic analysis in an attempt to discover serum markers that can assist in the early detection of HBV-induced liver cancer. Briefly, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation, as a function of cancer diagnosis, is described. The model we have used is the woodchuck (Marmota monax), which shares similarities in the glycosylation pattern associated with liver proteins in human HCC. In this report, we show that woodchucks diagnosed with HCC have dramatically higher levels of serum-associated core alpha-1,6-linked fucose, as compared with woodchucks without a diagnosis of HCC. The coupling of this methodology with 2D gel proteomics has permitted the identification of several glycoproteins with altered glycosylation as a function of cancer. One such glycoprotein, Golgi Protein 73 (GP73), was found to be elevated and hyperfucosylated in animals with HCC. Further, the study showed GP73 to be elevated in the serum of people with a diagnosis of HCC, providing a validation of our approach. The potential of this technology for biomarker discovery and the implications of increased levels of GP73 in liver cancer are discussed.

Animals↗
PubMed2005

Proteome analysis of a human uveal melanoma primary cell culture by 2-DE and MS.

We present here the first proteomics analysis of uveal melanoma (UM) cells. These cells represent a good model for the identification of polypeptide markers, which could be developed as diagnostic tools. UM is the most common primary intraocular tumour in adults. In contrast to other cancers, the survival rate of patients with this malignancy has changed little over the past few decades; a better understanding of the molecular biology of UM oncogenesis and metastasis is needed to build the basis for the identification of novel drug targets. In the study presented here, proteins from a UM primary cell culture were separated by 2-DE using a pI 3-10 gradient; 270 spots were analysed by LC-MS/MS, identifying 683 proteins derived from 393 different genes. Of those, 69 (18%) are related to cancer processes involving cell division, proliferation, invasion, metastasis, oncogenesis, drug resistance and others. To our knowledge, 96% of the proteins identified, including 16 hypothetical proteins, have never been reported in UM before. This study represents the first step towards the establishment of a UM protein database as a valuable resource for the study of this malignancy.

Biomarkers, Tumor↗
PubMed2005

Molecular mechanism of lipopeptide presentation by CD1a.

CD1a is expressed on Langerhans cells (LCs) and dendritic cells (DCs), where it mediates T cell recognition of glycolipid and lipopeptide antigens that contain either one or two alkyl chains. We demonstrate here that CD1a-restricted T cells can discriminate the peptide component of didehydroxymycobactin lipopeptides. Structure analysis of CD1a cocrystallized with a synthetic mycobactin lipopeptide at 2.8 A resolution further reveals that the single alkyl chain is inserted deep within the A' pocket of the groove, whereas its two peptidic branches protrude along the F' pocket to the outer, alpha-helical surface of CD1a for recognition by the TCR. Remarkably, the cyclized lysine branch of the peptide moiety lies in the shallow F' pocket in a conformation that closely mimics that of the alkyl chain in the CD1a-sulfatide structure. Thus, this structural study illustrates how a single chain lipid can be presented by CD1 and that the peptide moiety of the lipopeptide is recognized by the TCR.

Antigen Presentation↗
PubMed2005

Storage solutions: treating lysosomal disorders of the brain.

Many neurodegenerative diseases are characterized by the accumulation of undegradable molecules in cells or at extracellular sites in the brain. One such family of diseases is the lysosomal storage disorders, which result from defects in various aspects of lysosomal function. Until recently, there was little prospect of treating storage diseases involving the CNS. However, recent progress has been made in understanding these conditions and in translating the findings into experimental therapies. We review the developments in this field and discuss the similarities in pathological features between these diseases and some more common neurodegenerative disorders.

Animals↗
PubMed2005

Hepatitis B virus large and middle glycoproteins are degraded by a proteasome pathway in glucosidase-inhibited cells but not in cells with functional glucosidase enzyme.

The secretion of hepatitis B virus (HBV) large (LHBs) and middle (MHBs) envelope polypeptides from tissue cultures requires proper protein folding and is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase. Using competitive inhibitors of the ER glucosidase, here it is shown that the amounts of glycosylated and unglycosylated forms of LHBs and MHBs proteins are all greatly reduced in tissue cultures producing HBV envelope glycoproteins. In contrast, the HBV small (SHBs) protein was not affected. The reduction in secretion of LHBs and MHBs proteins appears to be mediated by proteasomal degradation pathways, since it is prevented by either lactacystin or epoxomicin, two inhibitors of proteasomal degradation. Although there is no detectable proteasomal degradation of LHBs and MHBs in cells with functional glucosidase, the implications of the nearly quantitative sensitivity of glycosylated and unglycosylated forms of LHBs and MHBs proteins, with selective sparing of SHBs protein, in cells in which glucosidase is inhibited is surprising, and its implications are discussed.

Acetylcysteine↗
PubMed2004

Alkylated imino sugars, reversible male infertility-inducing agents, do not affect the genetic integrity of male mouse germ cells during short-term treatment despite induction of sperm deformities.

Reversible infertility can be induced in male mice by oral administration of the alkylated imino sugars N-butyldeoxynojirimycin (NB-DNJ) and N-butyldeoxygalactonojirimycin (NB-DGJ). Spermatozoa of these mice have grossly misshapen heads and reduced motility. Because NB-DNJ and related compounds may hold promise as nonhormonal male contraceptives, a comprehensive examination of their effects on male reproduction is necessary. To this end, we further examined reproductive properties of the dysmorphic spermatozoa that are produced after short-term imino sugar administration at the minimal dose that completely abolishes the ability of male C57BL/6 mice to produce offspring by natural mating. Here, we report that, in vitro, the abnormal spermatozoa from the NB-DNJ- and NB-DGJ-treated mice were unable to fertilize oocytes. In addition, we investigated whether the imino sugars damage the genetic integrity of spermatozoa. To test this, we microsurgically injected deformed spermatozoa from imino sugar-treated males into oocytes. The deformed spermatozoa from the testis were able to activate oocytes very efficiently, but those from the cauda epididymis often failed to do so. This problem was overcome when the sperm-injected oocytes were treated with a parthenogenetic agent, Sr(2+). Oocytes injected with the misshapen spermatozoa from NB-DNJ- and NB-DGJ-treated mice developed (with or without Sr(2+) treatment) into live offspring that grew normally and were normally fertile. This indicates that during short-term administration, alkylated imino sugars alter sperm morphology and physiology but do not diminish the genetic potential of spermatozoa.

1-Deoxynojirimycin↗
PubMed2004

The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin.

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn(354) are inaccessible because the complex glycans at Asn(445) block access to the site. On IgE, the additional C(H)2 hinge domain blocks access to the oligomannose glycans at Asn(394) on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man(5)GlcNAc(2) glycans, which are the trimmed products of the Glc(3)Man(9)GlcNAc(2) oligomannose precursor. The presence of these structures suggests that the C(H)2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man(5)GlcNAc(2) during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.

Carbohydrate Conformation↗