Glycosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer

Human pancreatic ribonuclease 1 (RNase 1) is a glycoprotein expressed mainly by the pancreas and also found in endothelial cells. The diagnosis of pancreatic cancer (PaC) remains difficult and therefore the search for sensitive and specific markers is required. Previous studies showed that RNase 1 from human healthy pancreas contained only neutral glycans, whereas RNase 1 from PaC cell lines contained sialylated structures. To determine whether these glycan tumor cell-associated changes were also characteristic of serum RNase 1 and could be used as a marker of PaC, we have analyzed the glycosylation of serum RNase 1. The origin of serum RNase 1 was also investigated. Serum RNase 1 from two PaC patients and two controls was purified and the glycans analyzed by high-performance liquid chromatography (HPLC)-based sequencing and mass spectrometry. Although normal and tumor serum RNase 1 contained the same glycan structures, there was an increase of 40% in core fucosylation in the main sialylated biantennary glycans in the PaC serum RNase 1. This change in proportion would be indicative of a subset of tumor-associated glycoforms of RNase 1, which may provide a biomarker for PaC. Two-dimensional electrophoresis of the RNase 1 from several endothelial cell lines, EA.hy926, human umbilical vein endothelial cells (HUVEC), human mammary microvessel endothelial cells (HuMMEC), and human lung microvessel endothelial cells (HuLEC), showed basically the same pattern and was also very similar to that of serum RNase 1. RNase 1 from EA.hy926 was then purified and presented a glycosylation profile very similar to that from serum RNase 1, suggesting that endothelial cells are the main source of this enzyme.     

Pancreatic Cancer Cell Glycosylation Regulates Cell Adhesion and Invasion through the Modulation of α2β1 Integrin and E-Cadherin Function

In our previous studies we have described that ST3Gal III transfected pancreatic adenocarcinoma Capan-1 and MDAPanc-28 cells show increased membrane expression levels of sialyl-Lewis x (SLe^x) along with a concomitant decrease in α2,6-sialic acid compared to control cells. Here we have addressed the role of this glycosylation pattern in the functional properties of two glycoproteins involved in the processes of cancer cell invasion and migration, α2β1 integrin, the main receptor for type 1 collagen, and E-cadherin, responsible for cell-cell contacts and whose deregulation determines cell invasive capabilities. Our results demonstrate that ST3Gal III transfectants showed reduced cell-cell aggregation and increased invasive capacities. ST3Gal III transfected Capan-1 cells exhibited higher SLe^x and lower α2,6-sialic acid content on the glycans of their α2β1 integrin molecules. As a consequence, higher phosphorylation of focal adhesion kinase tyrosine 397, which is recognized as one of the first steps of integrin-derived signaling pathways, was observed in these cells upon adhesion to type 1 collagen. This molecular mechanism underlies the increased migration through collagen of these cells. In addition, the pancreatic adenocarcinoma cell lines as well as human pancreatic tumor tissues showed colocalization of SLe^x and E-cadherin, which was higher in the ST3Gal III transfectants. In conclusion, changes in the sialylation pattern of α2β1 integrin and E-cadherin appear to influence the functional role of these two glycoproteins supporting the role of these glycans as an underlying mechanism regulating pancreatic cancer cell adhesion and invasion.     

Ribonucleases expressed by human pancreatic adenocarcinoma cell lines.

Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N-glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N-glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker.     

Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG

Ovarian cancer is the fourth most common cancer in women in the Western world. In a pilot scale study, we highlight changes in the total serum glycome of patients with advanced ovarian cancer that might shed insight into disease pathogenesis. These changes include increases in levels of core fucosylated, agalactosyl biantennary glycans (FA2) and sialyl Lewis x (SLe(x)). To investigate further which proteins contribute to these alterations, we developed technology to analyze simultaneously the glycosylation of protein glycoforms contained in single spots excised from a 2D gel (<1 ng protein). The acute-phase proteins, haptoglobin, alpha1-acid glycoprotein, and alpha1-antichymotrypsin from patients contained elevated levels of subsets of glycoforms containing SLe(x). We also established that IgG heavy chains from patients contained twice the level of FA2 compared with healthy controls. Serum CA125 is the only biomarker that is used routinely, and there is a need for complementary markers that will improve both sensitivity and specificity. There was some preliminary indication that combinations of changes in the serum glycome might improve the separation of ovarian cancer and benign tumors; however, a larger study using data receiver operating characteristic curves will be required to draw any firm conclusions.     

Characterization of the glycosylation state of a recombinant monoclonal antibody using weak cation exchange chromatography and mass spectrometry.

Recombinant monoclonal antibody heterogeneity is inherent due to various enzymatic and non-enzymatic modifications. In this study, a recombinant humanized monoclonal IgG1 antibody with different states of glycosylation on the conserved asparagine residue in the CH(2) domain was analyzed by weak cation exchange chromatography. Two major peaks were observed and were further characterized by enzymatic digestion and mass spectrometry. It was found that this recombinant monoclonal antibody contained three glycosylation states of antibody with zero, one or two glycosylated heavy chains. The peak that eluted earlier on the cation exchange column contained antibodies with two glycosylated heavy chains containing fucosylated biantennary complex oligosaccharides with zero, one or two terminal galactose residues. The peak that eluted later from the column contained antibodies with either zero, one or two glycosylated heavy chains. The oligosaccharide on the antibodies eluted in the later peak was composed of only two GlcNAc residues. These results indicate that conformational changes in large proteins such as monoclonal antibodies, caused by different types of neutral oligosaccharides as well as the absence of oligosaccharides, can be differentiated by cation exchange column chromatography.     

Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins

Prostate-specific antigen (PSA) is a glycoprotein secreted by prostate epithelial cells. PSA is currently used as a marker of prostate carcinoma because high levels of PSA are indicative of a tumor situation. However, PSA tests still suffer from a lack of specificity to distinguish between benign prostate hyperplasia and prostate cancer. To determine whether PSA glycosylation could provide a means of differentiating between PSA from normal and tumor origins, N-glycan characterization of PSA from seminal fluid and prostate cancer cells (LNCaP cell line) by sequencing analysis and mass spectrometry was carried out. Glycans from normal PSA (that correspond to low and high pI PSA fractions) were sialylated biantennary complex structures, half of them being disialylated in the low pI PSA fraction and mostly monosialylated in the high pI PSA. PSA from LNCaP cells was purified to homogeneity, and its glycan analysis showed a significantly different pattern, especially in the outer ends of the biantennary complex structures. In contrast to normal PSA glycans, which were sialylated, LNCaP PSA oligosaccharides were all neutral and contained a higher fucose content. In 10-15% of the structures fucose was linked alpha1-2 to galactose, forming the H2 epitope absent in normal PSA. GalNAc was increased in LNCaP glycans to 65%, whereas in normal PSA it was only present in 25% of the structures. These carbohydrate differences allow a distinction to be made between PSA from normal and tumor origins and suggest a valuable biochemical tool for diagnosis and follow-up purposes.     

Defective galactosylation of serum transferrin in galactosemia

The glycosylation of serum transferrin from galactosemic patients with a deficiency of galactose-1-phosphate uridyl transferase (EC 2. 7.7 12) is abnormal but becomes normal after treatment with a galactose-free diet. To understand the structural and biochemical basis of the abnormal glycosylation, transferrin was purified from the serum of untreated and treated galactosemic patients and normal controls and the N-linked glycans analyzed by HPLC. The glycans from normal transferrin consisted predominantly (86%) of the disialylated biantennary complex type. The glycans from untreated galactosemic patients were more heterogeneous and contained four major truncated glycans in addition to a smaller amount (13%) of the disialylated biantennary complex type. The truncated glycans were deficient in galactose and sialic acid and their structures were consistent with a decrease in galactosyltransferase activity in hepatocytes, the probable cells of origin of the transferrin. This is postulated to be due to direct inhibition of the galactosyltransferase activity by the accumulated galactose-1-phosphate or to an effect on the formation of UDP- galactose, the donor substrate in the reaction. After treatment the proportion of the truncated glycans decreased and the proportion of the disialylated biantennary complex type increased, returning almost but never completely to normal, even after prolonged treatment in some cases. There was no clear relationship between the length of treatment and the normalization of glycosylation and the level of galactose-1- phosphate in red blood cells, the usual parameter for monitoring the treatment of galactosemics. It is suggested that the persistence of abnormally glycosylated proteins may contribute to the long-term complications in galactosemia.      

Controlled glycosylation of therapeutic antibodies in plants

Recombinant therapeutic monoclonal antibodies (mAb) can be expressed, assembled, and glycosylated in plants. Transgenic plants, producing anti-rabies mAb and anti-colorectal cancer mAb, were obtained from Agrobacterium-mediated transformation. The heavy chain (HC) of anti-rabies mAb was fused to the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal whereas the HC of anti-colorectal cancer mAb was not fused to the KDEL sequence. Gel release of glycans and detection by high-performance liquid chromatography (HPLC), together with computer assisted analysis and matrix-assisted laser desorption/ionization time-of-flight (MALD-TOF) mass spectrometry, revealed that the plant-derived anti-rabies mAb with KDEL contained mainly oligomannose type N-glycans while the plant-derived anti-colorectal cancer mAb carried mainly biantennary glycans with and without a pentose sugar, that is thought to be xylose. This finding indicates that the KDEL sequence can affect the N-glycosylation processing of antibody in plant cells. The plant-derived mAbs with addition of a KDEL sequence did not contain any of the known antigenic glycan epitopes that are frequently found in other plant glycans or in mammalian-derived mAbs. The altered glycosylation on both plant-derived mAbs did not affect the activities that are required for therapy. These results indicate that plant genetic engineering could provide an effective and inexpensive means to control the glycosylation of therapeutic proteins such as mAbs, by the addition of a KDEL signal as a regulatory element.     

Monoclonal antibodies against 5'-nucleotidase from a human pancreatic tumor cell line: their characterization and inhibitory capacity on tumor cell adhesion to fibronectin substratum.

Four mouse monoclonal antibodies (PTN63, PTN108, PTN124, PTN514) against the ecto-5'-nucleotidase purified from a human pancreatic adenocarcinoma cell line (PaTu II) have been raised and characterized. All four monoclonal antibodies recognize the protein moiety of the glycosylated ecto-5'-nucleotidase. In competition assays it was demonstrated that three of the antibodies (PTN63, PTN108, PTN514) recognize different epitopes within the protein moiety. Furthermore, PTN108, PTN124, and PTN514 reduced the 5'-nucleotidase AMPase activity in contrast to PTN63 having no inhibitory effect. The antibodies show no cross-reactivity with ecto-5'-nucleotidases from rat liver, bull seminal plasma, chicken gizzard and human peripheral blood cells. When assayed by indirect immunofluorescence the antibodies react with the plasma membrane of human pancreatic tumor cells with varying staining intensity. Immunocytochemistry on paraffin sections of normal human pancreas revealed a prominent staining of the pancreatic duct cells. No staining of the acinar and islet cells could be detected. Thus, 5'-nucleotidase is a marker enzyme for pancreatic duct cells and can be used to determine the origin of pancreatic tumor cells. PTN63 reduced the attachment to fibronectin substratum of a human pancreatic adenocarcinoma tumor cell line possessing a high amount of plasma membrane bound ecto-5'-nucleotidase, but had no effect on a cell line lacking the membrane bound AMPase. In contrast, PTN108 and PTN514, which inhibit the AMPase activity, exhibited no influence on the adhesion of human pancreatic tumor cells to fibronectin substratum.     

Membrane glycoproteomics of fetal lung fibroblasts using LC/MS

Some aberrant N‐glycosylations are being used as tumor markers, and glycoproteomics is expected to provide novel diagnosis markers and targets of drug developments. However, one has trouble in mass spectrometric glycoproteomics of membrane fraction because of lower intensity of glycopeptides in the existence of surfactants. Previously, we developed a glycopeptide enrichment method by acetone precipitation, and it was successfully applied to human serum glycoproteomics. In this study, we confirmed that this method is useful to remove the surfactants and applicable to membrane glycoproteomics. The glycoproteomic approach to the human fetal lung fibroblasts membrane fraction resulted in the identification of over 272 glycoforms on 63 sites of the 44 glycoproteins. According to the existing databases, the structural features on 41 sites are previously unreported. The most frequently occurring forms at N‐glycosylation site were high‐mannose type containing nine mannose residues (M9) and monosialo‐fucosylated biantennary oligosaccharides. Several unexpected N‐glycans, such as fucosylated complex‐type and fucosylated high‐mannose and/or fucosylated pauci‐mannose types were found in ER and lysosome proteins. Our method provides new insights into transport, biosynthesis, and degradation of glycoproteins.     

Analysis of the site-specific N-glycosylation of beta1,6 N-acetylglucosaminyltransferase V

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a beta1,6-linked GlcNAc to the alpha1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V.     

Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two-dimensional 1H n.m.r. and methylation analysis.

The structures of the oligosaccharides of the hemagglutinin of fowl plague virus [influenza A/FPV/Rostock/34 (H7N1)] have been elucidated by one- and two-dimensional 1H n.m.r. spectroscopy at 500 MHz and by microscale methylation analysis. N-Glycosidic oligosaccharides of the oligomannosidic (OM) and of the N-acetyllactosaminic type have been found, the latter type comprising biantennary structures, without (A) or with (E) bisecting N-acetylglucosamine, and triantennary (C) structures. Analysis of the tryptic and thermolytic glycopeptides of the hemagglutinin allowed the allocation of these oligosaccharides to the individual glycosylation sites. Each attachment site contained a unique set of oligosaccharides. Asn12 contains predominantly structures C and E which are highly fucosylated. Asn28 contains OM and A structures that lack fucose and sulfate. Asn123 shows A that has incomplete antennae but is highly fucosylated and sulfated. Asn149 has fucosylated A and E. Asn231 shows fucosylated A and E with incomplete antennae. Asn406 has OM oligosaccharides. Asn478 has A and E with little fucose. Localization of the oligosaccharides on the three-dimensional structure of the hemagglutinin revealed that the oligomannosidic glycans are attached to glycosylation sites at which the enzymes responsible for carbohydrate processing do not have proper access. These observations demonstrate that an important structural determinant for the oligosaccharide side chains is the structure of the glycoprotein itself. In addition, evidence was obtained that the rate of glycoprotein synthesis also has an influence on carbohydrate structure.     

Immunohistochemical localization of glucagon-like peptide 1. Use of poly- and monoclonal antibodies

We report the use of poly- and monoclonal antibodies to study the immunohistochemical distribution of glucagon-like peptide-1 immunoreactivity (GLP-1-IR) in various tissues. The polyclonal antibodies against GLP-1 reacted with pancreatic A cells, enteroglucagon (L) cells in the gut, and some neurons in the central nervous system of all species tested. In pancreas and gut the monoclonal antibodies against GLP-1 exhibited a similar, but species specific distribution, relative to the polyclonal antibodies. The colocalization of GLP-1 and glucagon immunoreactivity in pancreatic, intestinal, and nervous tissues is in agreement with previously reported findings that both peptides are part of a single precursor molecule (preproglucagon).     

Production and some properties of monoclonal antibodies against human pancreatic secretory trypsin inhibitor.

Hybridomas that secrete monoclonal antibodies against human pancreatic secretory trypsin inhibitor (PSTI) were established by fusion of spleen cells obtained from mice immunized with PSTI with mouse NS-I-Ag 4/1 myeloma cells. One of three resulting monoclonal antibodies (KN-1) was found to recognize the N-terminal moiety of the inhibitor, while the others (KN-2 and KN-3) reacted with other as yet undefined parts of the molecule. Trypsin inhibitory activity of PSTI treated with KN-1 monoclonal antibody was the same as that of PSTI itself, thus indicating no relationship between the N-terminal moiety of the PSTI molecule and its inhibitory activity. We further examined the applicability of one of the monoclonal antibodies (KN-1) for immunohistochemical study of human pancreatic cancer tissue including the normal as a model, and found granular staining of the cytoplasm of the normal acinar and duct cells and also of that of adenocarcinoma cells in formalin-fixed, paraffin-embedded tissue sections.     

Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.     

Various stages of schistosoma express Lewis(x), LacdiNAc, GalNAcbeta1-4 (Fucalpha1-3)GlcNAc and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc carbohydrate epitopes: detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins

We report here that fucosylated epitopes such as Lewis(x), LacdiNAc, fucosylated LacdiNAc (LDN-F) and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) are expressed by schistosomes throughout their life cycle. These four epitopes were enzymatically synthesized and coupled to bovine serum albumin to yield neoglycoproteins. Subsequently these neoglycoproteins were used to probe a panel of 188 monoclonal antibodies obtained from infected or immunized mice, in ELISA and surface plasmon resonance analysis. Of these antibodies, 25 recognized one of the fucosylated structures synthesized, indicating that these structures are immunogenic during infection. The MAbs identified could be subdivided in four different groups based on the recognition of either the Lewis(x)-, the LacdiNAc-, the LDN-DF-, or both the LDN-F- and LDN-DF epitope. These monoclonal antibodies were then used to investigate the localization of the fucosylated epitopes in various stages of Schistosoma mansoni using indirect immunofluorescence. Lewis(x)epitopes were mainly found in the gut and on the tegument of adult worms, on egg shells, and on the oral sucker of cercariae. The LacdiNAc epitope was expressed on the tegument of adult worms, on miracidia, and on the oral sucker of cercariae. In contrast, LDN-DF epitopes were mainly present in the excretory system of adult worms, on miracidia and on whole cercariae. These also stained positive with the LDN-F/LDN-DF epitope antibodies, while whole parenchyma reacted characteristically only with the latter antibodies. The identification of different carbohydrate structures in various stages of schistosomes may lead to a better understanding of the function of glycans in the immune response during infection.     

Immunohistochemical localization of glucagon-like peptide 1

Summary We report the use of poly-and monoclonal antibodies to study the immunohistochemical distribution of glucagon-like peptide-1 immunoreactivity (GLP-1-IR) in various tissues. The polyclonal antibodies against GLP-1 reacted with pancreatic A cells, enteroglucagon (L) cells in the gut, and some neurons in the central nervous system of all species tested. In pancreas and gut the monoclonal antibodies against GLP-1 exhibited a similar, but species specific distribution, relative to the polyclonal antibodies. The colocalization of GLP-1 and glucagon immunoreactivity in pancreatic, intestinal, and nervous tissues is in agreement with previously reported findings that both peptides are part of a single precursor molecule (preproglucagon).Supported by the DFG, SFB 90     

Modification of a recombinant GPI-anchored metalloproteinase for secretion alters the protein glycosylation

The N-linked glycans of recombinant leishmanolysin (GP63) expressed as a glycosylphosphatidylinositol (GPI)-anchored membrane protein or modified for secretion in Chinese hamster ovary (CHO) cells were analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The glycans isolated from both membrane and secreted protein were predominantly complex biantennary structures. However other aspects of the glycan profiles showed striking differences. The degree of sialylation of the membrane form was greatly reduced and the core fucosylation of biantennary structures was increased compared to the secreted form. Glycans isolated from membrane expressed protein also contained a higher proportion of lactosamine repeats. Residence times in the secretory pathway were similar for both secreted and membrane protein. Glycosylation differences may therefore be due to differences in protein conformation and accessibility to glycosyltransferases or glycosidases. These differences in glycosylation represent an important factor when considering modifying membrane expressed proteins for secreted production.     

Localization of defined carbohydrate epitopes in bovine polysialylated NCAM

Polysialylated neural cell adhesion molecule (NCAM) was immunoaffinity-purified from the brains of newborn calves. A degree of polymerization of up to 40 was chromatographically determined for released polysialic acid (PSA) chains. For characterization of N-glycan structures and attachment sites, PSA-NCAM was digested with trypsin, and the generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specific for PSA or the HNK1 epitope, i.e., HSO(3)-3GlcA(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-, yielding PSA-glycopeptides, HNK-glycopeptides and non-PSA/HNK1-(glyco) peptides. Using a combination of enzymatic deglycosylation, peptide fractionation, mass spectrometry and Edman degradation, HNK1-N-glycans could be assigned to glycosylation sites 2, 4, 5 and 6. Non-PSA/HNK1-glycans were assigned to glycosylation site 2, whereas PSA-N-glycans of bovine NCAM had been already previously shown to be restricted to glycosylation sites 5 and 6 (Glycobiology 12 (2002) 47). Respective oligosaccharides were enzymatically released, labeled with 2-aminopyridine and characterized by linkage analysis and mass spectrometry. Carbohydrate chains bearing PSA or the HNK1 epitope comprised mainly fucosylated, partially sulfated diantennary, triantennary or tetraantennary glycans without bisecting GlcNAc or fucosylated diantennary and triantennary species carrying, in part, bisecting GlcNAc residues, respectively. Some N-glycans simultaneously contained both the HNK1-epitope and PSA. Non-PSA/HNK1-glycans exhibited a heterogeneous pattern of partially truncated, mostly diantennary structures with one to three fucose residues, bisecting GlcNAc and/or sulfate residues. In addition, they were demonstrated to carry, to some extent, the Lewis X epitope. When compared with previous data on murine NCAM glycosylation, our results indicate a conservation of structural features and attachment sites for the different types of NCAM N-glycans.     

The glycan processing and site occupancy of recombinant Thy-1 is markedly affected by the presence of a glycosylphosphatidylinositol anchor

Thy-1 is a cell surface glycoprotein containing three N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The effect of the anchor on its N-linked glyco-sylation was investigated by comparing the glycosylation of soluble recombinant Thy-1 (sThy-1) with that of recombinant GPI anchored Thy-1, both expressed in Chinese hamster ovary cells. The sThy-1 was produced in a variety of isoforms including some which lacked carbohydrate on all three sequons whereas the GPI anchored form appeared fully glycosylated like native Thy-1. This was surprising as the attachment of N-linked sugars occurs cotranslationally and it was not expected that the presence of a C-terminal GPI anchor signal sequence would affect sequon occupancy. Furthermore sThy-1 lacking glycosylation could be produced with the inhibitor tunicamycin but in contrast cell surface expression of unglycosylated GPI anchored Thy-1 could not be obtained. The GPI anchored form appeared less processed with almost 4-fold more oligo-mannose oligosaccharides than in sThy-1 and also with less sialylated and core fucosylated biantennary glycans. Possible mechanisms whereby the anchor or the anchor signal sequence, control site occupancy and maturation are discussed.