Harald S. Conradt

Purification and Chemical Characterization of β-Trace Protein from Human Cerebrospinal Fluid: Its Identification as Prostaglandin D Synthase

Abstract: β‐Trace protein from pooled human CSF was purified to homogeneity. An apparent molecular mass of 23–29 kDa was determined for the polypeptide on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Amino‐terminal sequencing of the polypeptide yielded the unique amino acid sequence APEAQVSVQPNFQQDKFLGRWFSA24. Alignment of amino acid sequences obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identification of β‐trace protein as prostaglandin D synthase [prostaglandin‐H2 D‐isomerase; (5Z, 13E)‐(15S)‐9α, 11 a‐epidioxy‐15‐hydroxyprosta‐5,13‐dienoate D‐isomerase; EC 5.3.99.2]. A conservative amino acid exchange (The instead of Ser) was detected at amino acid position 154 of the β‐trace polypeptide chain in the corresponding tryptic peptide. The two N‐glycosylation sites of the polypeptide were shown to be almost quantitatively occupied by carbohydrate. Carbohydrate compositional as well as methylation analysis indicated that Asn29and Asn56 bear exclusively complex‐type oligosaccharide structures (partially sialylated with α2–3‐ and/or α2–6‐linked N‐acetylneuraminic acid) that are almost quantitatively α1‐6 fucosylated at the proximal N‐acetylglucosamine; ∼70% of these molecules contain a bisecting N‐acetylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.

GENETIC ENGINEERING OF RECOMBINANT GLYCOPROTEINS AND THE GLYCOSYLATION PATHWAY IN MAMMALIAN HOST CELLS

The analysis of many natural glycoproteins and their recombinant counterparts from mammalian hosts has revealed that the basic oligosaccharide structures and the site occupancy of glycosylated polypeptides are primarily dictated by the protein conformation.

The Cytoplasmic, Transmembrane, and Stem Regions of Glycosyltransferases Specify Their in Vivo Functional Sublocalization and Stability in the Golgi

We provide evidence for the presence of targeting signals in the cytoplasmic, transmembrane, and stem (CTS) regions of Golgi glycosyltransferases that mediate sorting of their intracellular catalytic activity into different functional subcompartmental areas of the Golgi. We have constructed chimeras of human α1,3-fucosyltransferase VI (FT6) by replacement of its CTS region with those of late and early acting Golgi glycosyltransferases and have stably coexpressed these constructs in BHK-21 cells together with the secretory reporter glycoprotein human β-trace protein. The sialyl Lewis X:Lewis X ratios detected in β-trace protein indicate that the CTS regions of the early acting GlcNAc-transferases I (GnT-I) and III (GnT-III) specify backward targeting of the FT6 catalytic domain, whereas the CTS region of the late acting human α1,3-fucosyltransferase VII (FT7) causes forward targeting of the FT6in vivo activity in the biosynthetic glycosylation pathway. The analysis of the in vivo functional activity of nine different CTS chimeras toward β-trace protein allowed for a mapping of the CTS donor glycosyltransferases within the Golgi/trans-Golgi network: GnT-I < (ST6Gal I, ST3Gal III) < GnT-III < ST8Sia IV < GalT-I < (FT3, FT6) < ST3Gal IV < FT7. The sensitivity or resistance of the donor glycosyltransferases toward intracellular proteolysis is transferred to the chimeric enzymes together with their CTS regions. Apparently, there are at least three different signals contained in the CTS regions of glycosyltransferases mediating: first, their Golgi retention; second, their targeting to specific in vivofunctional areas; and third, their susceptibility toward intracellular proteolysis as a tool for the regulation of the intracellular turnover.

Identification of the human Lewisa carbohydrate motif in a secretory peroxidase from a plant cell suspension culture (Vaccinium myrtillus L.)

This paper reports for the first time the presence of the human Lewisa type determinant in glycoproteins secreted by plant cells. A single glycopeptide was identified in the tryptic hydrolysis of the peroxidase VMPxC1 from Vaccinium myrtillus L. by HPLC/ESI‐MS. The oligosaccharide structures were elucidated by ESI‐MS‐MS and by methylation analysis before and after removal of fucose by mild acid hydrolysis. The major structure determined is of the biantennary plant complex type containing the outer chain motif Lewisa A corresponding fucosyltransferase activity catalyzing the formation of Lewisa type structures in vitro was identified in cellular extracts of the suspension cultures.

Characterization of changes in the glycosylation pattern of recombinant proteins from BHK-21 cells due to different culture conditions

The N-glycosylation patterns of a genetically engineered human interleukin-2 variant glycoprotein (IL-Mu6), produced by BHK-21 cells from long-term suspension and microcarrier cultures in the presence and absence of fetal calf serum were compared. IL-Mu6 was used as a model protein in studying the effect of different controlled cell culture conditions on the expression of N-glycans in recombinant glycoproteins. IL-Mu6 contains a single amino acid substitution (Glu100<==>Asn) generating a potential N-glycosylation recognition site (Asn100-Xxx-Thr/Ser) in addition to the natural O-glycosylation at position Thr3. Parallel cell cultivations were carried out in two continuously perfused 2.5-liter stirred bioreactors under defined culture conditions. Major differences were found in the glycoprotein products obtained during these different cultivation conditions. Serum-free cultures resulted in a higher level of terminal sialylation and proximal alpha 1-6 fucosylation. The ratio of O- to N-glycans as well as the amount of nonglycosylated product and the antennarity of N-linked carbohydrates in the model protein exhibited major differences depending on the presence or absence of serum, the condition of growth and the cultivation procedure.

Temperature reduction in cultures of hGM-CSF-expressing CHO cells: effect on productivity and product quality.

We have demonstrated that temperature reduction from 37 to 33 °C in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO‐K1‐hGM‐CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO‐K1‐hGM‐CSF cells were cultured in a biphasic mode: first, a 37 °C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 °C. The maximum cell density was not affected after temperature reduction while cell viability remained above 80% for a further 3.7 days in the culture kept at the lower temperature, when compared to the control culture maintained at 37 °C. Furthermore, the total rhGM‐CSF production increased 6 times in the culture shifted to 33 °C. Because the quality and hence the in vivo efficacy of a recombinant protein might be affected by numerous factors, we have analyzed the N‐ and O‐glycosylation of the protein produced under both cell culture conditions using high‐pH anion‐exchange chromatography and complementary mass spectrometry techniques. The product quality data obtained from the purified protein preparations indicated that decreasing temperature had no significant effect on the rhGM‐CSF glycosylation profiles, including the degree of terminal sialylation. Moreover, both preparations exhibited the same specific in vitro biological activity. These results revealed that the employed strategy had a positive effect on the cell specific productivity of CHO‐K1‐hGM‐CSF cells without affecting product quality, representing a novel procedure for the rhGM‐CSF production process.

In Vivo Specificity of Human α1,3/4-Fucosyltransferases III-VII in the Biosynthesis of LewisX and Sialyl LewisX Motifs on Complex-type N-Glycans COEXPRESSION STUDIES FROM BHK-21 CELLS TOGETHER WITH HUMAN β-TRACE PROTEIN

Each of the five human α1,3/4-fucosyltransferases (FT3 to FT7) has been stably expressed in BHK-21 cells together with human β-trace protein (β-TP) as a secretory reporter glycoprotein. In order to study their in vivo properties for the transfer of peripheral Fuc ontoN-linked complex-type glycans, detailed structural analysis was performed on the purified glycoprotein. All fucosyltransferases were found to peripherally fucosylate 19–52% of the diantennary β-TP N-glycans, and all enzymes were capable of synthesizing the sialyl LewisX (sLex) motif. However, each enzyme produced its own characteristic ratio of sLex/Lex antennae as follows: FT7 (only sLex), FT3 (14:1), FT5 (3:1), FT6 (1.1:1), and FT4 (1:7). Fucose transfer onto β-TP N-glycans was low in FT3 cells (11% of total antennae), whereas the values for FT7, FT5, FT4, and FT6 cells were 21, 25, 35, and 47%, respectively. FT3, FT4, FT5, and FT7 transfer preponderantly one Fuc per diantennary N-glycan. FT4 preferentially synthesizes di-Lex on asialo diantennaryN-glycans and mono-Lex with monosialo chains. In contrast, FT6 forms mostly α1,3-difucosylated chains with no, one, or two NeuAc residues. FT3, FT4, and FT6 were proteolytically cleaved and released into the culture medium in significant amounts, whereas FT7 and FT5 were found to be largely resistant toward proteolysis. Studies on engineered soluble variants of FT6 indicate that these forms do not significantly contribute to the in vivo fucose transfer activity of the enzyme when expressed at activity levels comparable to those obtained for the wild-type Golgi form of FT6 in the recombinant host cells.

Sequencing of tri- and tetraantennary N-glycans containing sialic acid by negative mode ESI QTOF tandem MS

Application of the negative mode electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI QTOF) tandem MS for determination of substitution patterns by sialic acid and/or fucose and extention by additional LacNAc disaccharide units in single branches of multiantennary N-glycans from biological samples is described. Fragmentation patterns which can be obtained by low energy collision-induced dissociation (CID) using the QTOF instrument include cleavage ions, diagnostic for determination of antennarity and for specific structural features of single antennae. Systematic fragmentation studies in the negative ion mode were focussed toward formation of the D diagnostic ion relevant for assignment of 3- and 6-antennae in complex N-glycans carrying three and four antennae in combination with epitope-relevant B- and C-type ions. For validation of this approach ESI QTOF fragmentation of the permethylated analogues was carried out in the positive ion mode. Using this strategy, products of in vitro glycosylation reactions were investigated in order to clarify some general aspects of N-glycan acceptor specificity during biosynthesis. α1-3fucosylation using GDP-fucose along with a soluble form of the recombinant human α1-3fucosyltransferase VI was carried out on tri- and tetraantennary precursors to test structural requirements for formation of Lex versus sLex motifs.

Carbohydrate Structures of β-Trace Protein from Human Cerebrospinal Fluid: Evidence for “Brain-Type”N-Glycosylation

Abstract: The carbohydrate structures of β‐trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N‐linked oligosaccharides at two sites (Asn29 and Asn56). Enzymatically released N‐glycans were studied by compositional and methylation analyses, high‐pH anion‐exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N‐acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N‐acetylglucosamine residues of nearly all structures were found to be α1,6‐fucosylated. Peripheral fucose (about 20%α1,3‐linked to N‐acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N‐acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N‐acetylglucosamine only were present. At least 20 different N‐glycans were identified. Analysis of the site‐specific glycosylation patterns at Asn29 and Asn56 revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N‐acetylglucosamine, as well as terminal N‐acetylglucosamine and galactose residues, and significant amounts of N‐acetylneuraminic acid in α2,3 linkage), this protein can be classified as “brain‐type” glycosylated.

'BRAIN-TYPE' N-GLYCOSYLATION OF ASIALO-TRANSFERRIN FROM HUMAN CEREBROSPINAL FLUID

Asiolo‐transferrin from human cerebrospinal fluid was purified to homogeneity. Investigation of the structural characteristics of its oligosaccharides support our hypothesis of ‘brain‐type’ glycosylation of intrathecally synthesized cerebrospinal fluid proteins. For carbohydrate structural analysis, high‐pH anion‐exchange chromatography, methylation analysis, liquid secondary ion‐ and matrix‐assisted laser desorption/ ionization mass spectrometry of the permethylated derivatives were used. The major structure turned out to be a complex‐type agalacto‐diantennary oligosaccharide with bisecting N‐acetylglucosamine and proximal fucose. Analysis of a second transferrin preparation containing both asialo‐ and sialo‐transferrin revealed another major glycan species derived from the sialylated transferrin variant which is galactosylated and lacks bisecting N‐acetylglucosamine and fucose.