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H Clausen

Publications and source records attributed to H Clausen.

At least 73 records · Page 4Linked to original sources
PubMed1996

cDNA cloning and expression of a novel human UDP-N-acetyl-alpha-D-galactosamine. Polypeptide N-acetylgalactosaminyltransferase, GalNAc-t3.

The glycosylation of serine and threonine residues during mucin-type O-linked protein glycosylation is carried out by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferase). Previously two members, GalNAc-T1 and -T2, have been isolated and the genes cloned and characterized. Here we report the cDNA cloning and expression of a novel GalNAc-transferase termed GalNAc-T3. The gene was isolated and cloned based on the identification of a GalNAc-transferase motif (61 amino acids) that is shared between GalNAc-T1 and -T2 as well as a homologous Caenorhabditis elegans gene. The cDNA sequence has a 633-amino acid coding region indicating a protein of 72.5 kDa with a type II domain structure. The overall amino acid sequence similarity with GalNAc-T1 and -T2 is approximately 45%; 12 cysteine residues that are shared between GalNAc-T1 and -T2 are also found in GalNAc-T3. GalNAc-T3 was expressed as a soluble protein without the hydrophobic transmembrane domain in insect cells using a Baculo-virus vector, and the expressed GalNAc-transferase activity showed substrate specificity different from that previously reported for GalNAc-T1 and -T2. Northern analysis of human organs revealed a very restricted expression pattern of GalNAc-T3.

Amino Acid Sequence↗
PubMed1996

Histo-blood group p: biosynthesis of globoseries glycolipids in EBV-transformed B cell lines.

The genetic and biosynthetic basis of the histo-blood group P-system is not fully understood. Individuals with the rare p phenotype do not express the three glycolipid antigens (Pk, P and P1) of this system, probably because of deficiencies in glycosyltransferases involved in their biosynthesis. Iiuka et al. [Iiuka S, Chen SH, Yoshida A (1986) Biochem Biophys Res Commun 137: 1187-95], however, previously reported that detergent extracts from an EBV-transformed B cell line derived from a p individual did express the glycosyltransferase activity (Pk transferase) assumed to be missing in this blood group status. Here, we have reinvestigated the antigen expression and glycosyltransferase activities in two p individuals by analysing EBV-transformed cell lines as well as erythrocytes to confirm the blood group P status. The thin layer chromatography glycolipid profile of extracts from erythrocytes and EBV-transformed B cell lines showed characteristic accumulation of lactosylceramide and absence of Pk and P antigens. Glycosyltransferase activities of the B cell lines were analysed using glycolipid substrates and both extracts were found to contain lactosylceramide synthetase and P transferase activities but to be completely devoid of Pk transferase activity. The presented data indicate that p individuals, in contrast to previous reports, do not express a functional Pk glycosyltransferase.

Antigens, Surface↗
PubMed1996

Sensitivity of HIV-1 to neutralization by antibodies against O-linked carbohydrate epitopes despite deletion of O-glycosylation signals in the V3 loop.

It has been suggested that threonine or serine residues in the V3 loop of HIV-1 gp120 are glycosylated with the short-chain O-linked oligosaccharides Tn or sialosyl-Tn that function as epitopes for broadly neutralizing carbohydrate specific antibodies. In this study we examined whether mutation of such threonine or serine residues could decrease the sensitivity to infectivity inhibition by Tn or sialosyl-Tn specific antibodies. All potentially O-glycosylated threonine and serine residues in the V3 loop of cloned HIV-1 BRU were mutagenized to alanine thus abrogating any O-glycosylation at these sites. Additionally, one of these T-A mutants (T308A) also abrogated the signal for N-glycosylation at N306 inside the V3-loop. The mutant clones were compared with the wild type virus as to sensitivity to neutralization with monoclonal and polyclonal antibodies specific for the tip of the V3 loop of BRU or for the O-linked oligosaccharides Tn or sialosyl-Tn. Deletion of the N-linked oligosaccharide at N306 increased the neutralization sensitivity to antibodies specific for the tip of the loop, which indicates that N-linked glycosylation modulates the accessibility to this immunodominant epitope. However, none of the mutants with deletions of O-glycosylation signals in the V3 loop displayed any decrease in sensitivity to anti-Tn or anti-sialosyl-Tn antibody. This indicates that these broadly specific neutralization epitopes are located outside the V3 loop of gp 120.

Amino Acid Sequence↗
PubMed1996

Sorting by COP I-coated vesicles under interphase and mitotic conditions.

COP I-coated vesicles were analyzed for their content of resident Golgi enzymes (N-acetylgalactosaminyltransferase; N-acetylglucosaminyltransferase I; mannosidase II; galactosyltransferase), cargo (rat serum albumin; polyimmunoglobulin receptor), and recycling proteins (-KDEL receptor; ERGIC-53/p58) using biochemical and morphological techniques. The levels of these proteins were similar when the vesicles were prepared under interphase or mitotic conditions showing that sorting was unaffected. The average density relative to starting membranes for resident enzymes (14-30%), cargo (16-23%), and recycling proteins (81-125%) provides clues to the function of COP I vesicles in transport through the Golgi apparatus.

Amino Acid Sequence↗
PubMed1996

Psychological and social impact of carrier screening for cystic fibrosis among pregnant woman--a pilot study.

The aim of the current study was to assess the psychological and social impact of delta F508 carrier screening for cystic fibrosis among pregnant women. The impact of carrier screening was assessed in terms of anxiety, perception of health, reproductive decisions, retention of results, and sharing of information with relatives by two self-administered questionnaires sent to 160 women with a positive and 200 women with a negative test result. While no attempt was made to make women accept or decline testing, 22-28% of those tested found it difficult to reject the test when offered. Women with a positive test result became more anxious than did women with a negative result. However, their perception of future health did not change. Most carriers shared the information about their result with relatives and friends. Carriers had the best retention of pretest information and test result, although a third of the carriers believed that they could not have a child with cystic fibrosis when the partner's test for delta F508 and five other mutations were negative. Most women with a negative test result did not remember their result correctly a year after testing. Few women with a positive result changed their reproductive plans because of the result of the test.

Attitude to Health↗
PubMed1996

Psychological impact of carrier screening for cystic fibrosis among pregnant women.

The aim of the present study was to assess the impact of being identified carrier of cystic fibrosis. The impact was assessed in terms of retention of the result, sharing of the information about the result with relatives, non-relatives and GPs, changes in reproductive plans, and regrets about having been tested Three unsupervised questionnaires were sent to 160 women identified as carriers between 1990 and 1992 in June 1992, October 1993, and November 1994. Carriers freely shared the information about their result with relatives, friends, and GPs. The inconclusiveness of the test gave rise to some confusion. This may reflect inadequacies in the information and counselling given to carriers, but psychological factors are also believed to be responsible. Thus, false reassurance may be a problem in a carrier screening with a test that detect only a proportion of carriers. Few carriers considered changing their reproductive plans due to the result of the test. A few women identified as carrier regretted having had the test.

Cystic Fibrosis↗
PubMed1996

Differential expression of human high-molecular-weight salivary mucin (MG1) and low-molecular-weight salivary mucin (MG2).

Two distinct mucin components of saliva, MG1 and MG2, have been identified based on chemical composition and molecular weights (high and low, respectively) in saliva. With the aim of characterizing the expression pattern of salivary mucins, we have prepared monoclonal antibodies (MAbs) directed against the peptide core of MG1 and against a synthetic peptide derived from the MG2 (MUC7) sequence. MAb PANH2 raised against partially deglycosylated MG1 stained a high-molecular-weight smear in Western blots of partially purified MG1. PANH2 binding was increased by deglycosylation with trifluoromethanesulfonic acid as well as with subsequent periodate treatment, and was eliminated by pronase treatment, strongly suggesting that MAb PANH2 was directed to a peptide epitope of MG1. MAb PANH3 raised against a synthetic peptide derived from the MG2 (MUC7) sequence reacted with the native molecule and stained a narrow smear of ca. 200,000 to 210,000 in Western blots of concentrated saliva and a lower-molecular-weight smear of trifluoromethanesulfonic-acid-treated MG2. Immunohistology on frozen sections of human salivary glands showed that MAb PANH2 selectively labeled mucous cells, whereas MAb PANH3 labeled subpopulations of serous cells. Double-direct immunofluorescence staining with PANH2 and PANH3 demonstrated that the staining patterns were non-overlapping. The development of these antibody probes will facilitate studies of mucin expression in diseases of salivary glands.

Adult↗
PubMed1995

Purification and cDNA cloning of a human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (GalNAc-transferase) from human placenta was purified to apparent homogeneity using a synthetic acceptor peptide as affinity ligand. The purified GalNAc-transferase migrated as a single band with an approximate molecular weight of 52,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on a partial amino acid sequence, the cDNA encoding the transferase was cloned and sequenced from a cDNA library of a human cancer cell line. The cDNA sequence has a 571-amino acid coding region indicating a protein of 64.7 kDa with a type II domain structure. The deduced protein sequence showed significant similarity to a recently cloned bovine polypeptide GalNAc-transferase (Homa, F.L., Hollanders, T., Lehman, D.J., Thomsen, D.R., and Elhammer, A.P. (1993) J. Biol. Chem. 268, 12609-12616). A polymerase chain reaction construct was expressed in insect cells using a baculovirus vector. Northern analysis of eight human tissues differed clearly from that of the bovine GalNAc-transferase. Polymerase chain reaction cloning and sequencing of the human version of the bovine transferase are presented, and 98% similarity at the amino acid level was found. The data suggest that the purified human GalNAc-transferase is a novel member of a family of polypeptide GalNAc-transferases, and a nomenclature GalNAc-T1 and GalNAc-T2 is introduced to distinguish the members.

Amino Acid Sequence↗
PubMed1995

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Identification and separation of two distinct transferase activities.

Using a defined acceptor substrate peptide as an affinity chromatography ligand we have developed a purification scheme for a unique human polypeptide, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) (White, T., Bennett, E.P., Takio, K., Sørensen, T., Bonding, N., and Clausen, H. (1995) J. Biol. Chem. 270, 24156-24165). Here we report detailed studies of the acceptor substrate specificity of GalNAc-transferase purified by this scheme as well as the Gal-NAc-transferase activity, which, upon repeated affinity chromatography, evaded purification by this affinity ligand. Using a panel of acceptor peptides, a qualitative difference in specificity between these separated transferase activities in four rat organs and two human organs also revealed qualitative differences in specificity. The results support the existence of multiple Gal-NAc-transferase activities and suggest that these are differentially expressed in different organs. As the number of GalNAc-transferases existing is unknown, as is the specificity of the until now cloned and expressed GalNAc-transferases (T1 and T2), it is as yet impossible to relate the results obtained to specific enzyme proteins. The identification of acceptor peptides that can be used to discriminate GalNAc-transferase activities is an important step toward understanding the molecular basis of GalNAc O-linked glycosylation in cells and organs and in pathological conditions.

Amino Acid Sequence↗
PubMed1995

Gene expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat hypothalamus.

Pituitary adenylate cyclase activating polypeptide (PACAP) isolated from ovine hypothalamus is considered to be a member of the vasoactive intestinal peptide/glucagon/secretin/growth hormone-releasing hormone family of peptides. Two forms of PACAP, PACAP38 and PACAP27, have been demonstrated in the rat hypothalamus. The PACAP precursor contains another peptide called PACAP-related peptide (PRP), but so far no information on this peptide in tissue exists. We have developed three radioimmunoassays specific for PACAP38, PACAP27 and PRP and demonstrate that all three preproPACAP peptides are expressed in the rat hypothalamus, the PACAP38/PACAP27 ratio being around 60 and the PACAP38/PRP ratio being around 10. HPLC analysis of hypothalamic extract showed that PACAP38 and PACAP27 are found in only one form corresponding to the respective synthetic peptides, whereas PRP eluted in two peaks, the predominant form corresponding to synthetic PRP1-29. The cellular distribution of PACAP38, PACAP27, and PRP and corresponding mRNA in the hypothalamus was determined with immunohistochemistry and in situ hybridization histochemistry. PACAP- and PRP-immunoreactive neuronal perikarya were observed in the medial parvocellular part of the paraventricular nucleus (PVN) in colchicine pretreated rats. Some cell bodies of magnocellular variety were found in the PVN. PACAP mRNA containing cells were observed in moderate numbers in the same parts of the paraventricular nucleus. PACAP- and PRP immunoreactive fibres and varicosities were distributed in the PVN and in the periventricular nucleus. These data show that PACAP38, PACAP27 and PRP are expressed in the parvocellular part of the PVN, implying roles as hypothalamic regulatory peptides.

Animals↗
PubMed1995

Genomic cloning of the human histo-blood group ABO locus.

A P1 phage clone containing the near complete protein coding region of the human histo-blood group ABO gene locus was isolated and characterized. The insert was estimated at approximately 41 kp, and an approximately 20 kbp BamHI fragment contained the near complete coding region as evaluated by Southern blot analysis. Intron/exon boundaries were determined and the coding region was found to span at least seven exons. In situ hybridization to metaphase chromosomes confirmed the predicted localization to 9q34.

ABO Blood-Group System↗
PubMed1994

Human tumor-associated Le(a)-Le(x) hybrid carbohydrate antigen IV3(Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc)III3FucnLc4 defined by monoclonal antibody 43-9F: enzymatic synthesis, structural characterization, and comparative reactivity with various antibodies.

Two Le(a)-cross-reacting monoclonal antibodies (mAbs) were previously established which define complex tumor-associated carbohydrate antigens. The first mAb, 43-9F, was raised against human squamous cell lung carcinoma and shows preferential reactivity with various human cancers over normal cells. Its tumor cell binding activity is best inhibited by a milk oligosaccharide characterized as Le(a)-Le(x) [Mårtensson, S., et al. (1988) Cancer Res. 48, 2125], Gal beta 1-->3[Fuc alpha 1-->4] GlcNAc beta 1-->3Gal beta 1-->4]Fuc alpha 1-->3]-GlcNAc beta 1-->3Gal beta 1-->4Glc (1). The second mAb, ST-421, was raised against human gastric cancer xenograft in nude mice and found to have strong tumor growth-suppressing activity in nude mice. The epitope recognized by ST-421 was chemically identified as Le(a)-Le(a), Gal beta 1-->3 [Fuc alpha 1-->4]GlcNAc beta 1-->3Gal beta 1-->3-[Fuc alpha 1-->4] GlcNAc beta 1-->3Gal beta 1-->4Glc (2) [Stroud, M.R., et al. (1991) J. Biol. Chem. 266, 8439]. Both 43-9F and ST-421 cross-react with Le(a). Identification of the 43-9F antigen as structure 1 (Le(a)-Le(x)) is tentative since it was not based on isolation and chemical characterization of antigen from tumor cells or tissues. We therefore synthesized structure 1 starting from sialyl-nor-hexaosylceramide (VI3NeuAcnLc6), with sequential enzymatic hydrolysis by sialidase and beta-galactosidase followed by addition of beta 1-->3Gal with beta 1-->3 galactosyltransferase. This yielded the hybrid type 1/type 2 chain core structure IV3-(Gal beta 1-->3GlcNAc)nLc4, which was fucosylated with alpha 1-->3/4 fucosyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1994

Blood group ABO-related glycosylation of urothelial cell lines: immunocytological, enzymatic, and genetic characterization.

Three immortalized, human urothelial cell lines were characterized with respect to their ABO-related carbohydrate phenotypes using a panel of monoclonal antibodies directed to a series of carbohydrate epitopes (Lac, sialylated Lac, Le(a), sialylated Le(a), Le(x), sialylated Le(x), H types I and II, Ley, Leb, A monofucosylated types I and II, ALey, Aleb, and A type III). The glycosyltransferases forming some of these epitopes (beta 1-3/4 galactosyltransferase, alpha 1-2 fucosyltransferase, alpha 1-3 galactosyltransferase, and alpha 1-3-N-acetyl-galactosaminyltransferase) were determined by enzyme assays. The ABO gene complex was analyzed by Southern blotting, Northern blotting, and polymerase chain reaction across the O deletion and across base differences between the A and B alleles. The immunocytochemical stainings showed marked differences between the three cell lines; the high grade (tumorigenic, metastatic) cell line showed difucosylated types I and II structures, and the low grade (nontumorigenic, nonmetastatic) cell lines showed monofucosylated types I and II structures. Polymerase chain reaction genotyping of the cell lines indicated that one was OO, one was AA, and one was A plus a mutated allele. Northern blotting showed RNA encoding the A transferase. However, even though both of the A cell lines seemed to have an intact gene, which could produce A transferase and transcribed RNA, none of them showed any activity of the A gene encoded enzyme or any A-structures at the cell surface. In contrast, the three other examined glycosyltransferases were active. The three urothelial cell lines reflect in vivo findings in humans. They represent a competent system for in vitro studies of the different carbohydrate transferase genes responsible for the carbohydrate structures expressed on the cell surface in bladder tumors.

ABO Blood-Group System↗
PubMed1994

Localization of O-glycan initiation, sphingomyelin synthesis, and glucosylceramide synthesis in Vero cells with respect to the endoplasmic reticulum-Golgi intermediate compartment.

The identification of an endoplasmic reticulum-Golgi intermediate compartment (ERGIC), defined by the 53-kDa transmembrane marker protein ERGIC-53, has added to the complexity of the exocytic pathway of higher eukaryotic cells. Recently, a subcellular fractionation procedure was established for the isolation of the ERGIC from Vero cells (Schweizer, A., Matter, K., Ketcham, C. M., and Hauri, H.-P. (1991) J. Cell Biol. 113, 45-54) which provides a means to study more precisely the compartmentalization of the various enzymic functions along the early secretory pathway. Here, we have investigated if O-glycan initiation and sphingomyelin synthesis are associated with the ERGIC by analyzing both the responsible enzyme activities and their corresponding products. Moreover, the synthesis of glucosylceramide, the precursor of most glycosphingolipids, was also analyzed. In the purified ERGIC fraction UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc transferase) was only minimally enriched, sphingomyelin synthase was not enriched, and UDP-glucose:ceramide-glucosyl transferase specific activity was lower than in the homogenate. On Percoll gradients all three enzymes cofractionated with Golgi markers rather than ERGIC-53. Accordingly, sphingomyelin concentrations were extremely low in the ERGIC fraction. Double immunofluorescence localization of core N-acetylgalactosamine, the product of GalNAc transferase, by monoclonal antibodies against GalNAc-Ser/Thr (Tn antigen) revealed only little apparent overlap with ERGIC-53. This was particularly evident in brefeldin A-treated cells which showed entirely different patterns of Tn antigens and ERGIC-53. The results suggest that in the secretory pathway of Vero cells O-glycan initiation and sphingomyelin as well as glucosylceramide synthesis mainly occur beyond the ERGIC in the Golgi apparatus.

Amino Acid Sequence↗
PubMed1994

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells.

The mucin-type carbohydrate Tn cryptantigen (GalNAc alpha 1-O-Ser/Thr, where GalNAc is N-acetyl-D-galactosamine) is expressed in many carcinomas, in haemopoietic disorders including the Tn syndrome, and on human immunodeficiency virus (HIV) coat glycoproteins, but is not expressed on normal, differentiated cells because of the expression of a Tn-processing galactosyltransferase. Using Jurkat T leukaemic cells which express high levels of Tn antigen due to deficient Tn galactosylation, we have established the Tn antigen-mediated gene transfer and demonstrate the considerable efficiency of this approach. We used poly(L-lysine) conjugates of the monoclonal antibody 1E3 directed against the Tn antigen to deliver the luciferase and beta-galactosidase reporter genes to Jurkat cells by receptor-mediated endocytosis. Addition of unconjugated 1E3 reduced transfection efficiency in a concentration-dependent manner and incubation with free GalNAc abolished DNA transfer completely, indicating that gene delivery is indeed mediated by the Tn antigen. Pre-treatment of Jurkat cells with Vibrio cholerae sialidase, which uncovers additional Tn antigens, resulted in an improvement of gene transfection. Both human and chicken adenovirus particles attached to the DNA/polylysine complex strongly augmented transgene expression. When the beta-galactosidase (lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis, up to 60% of the cells were positive in the cytochemical stain using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a chromogenic substrate. The efficiency of the transferrin receptor-mediated DNA uptake into Jurkat cells was comparatively low, although these cells were shown to express considerable amounts of transferrin receptor. We show here that a mucin-type carbohydrate antigen mediates highly efficient DNA uptake by endocytosis into Jurkat T cells. This method represents a 50-fold improvement of Jurkat cell transfection efficiency over other physical gene transfer techniques. Specific gene delivery to primary cancer cells exhibiting Tn epitopes may especially be desirable in immunotherapy protocols.

Adenoviridae↗