Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins.

An HEK293S cell line resistant to ricin was prepared by mutagenesis by using ethyl methanesulfonate. It was shown to lack N-acetylglucosaminyltransferase I (GnTI) activity, and consequently unable to synthesize complex N-glycans. The tetracycline-inducible opsin expression system was assembled into this GnTI− HEK293S cell line. Stable cell lines were isolated that gave tetracycline/sodium butyrate-inducible expression of the WT opsin gene at levels comparable with those observed in the parent tetracycline-inducible HEK293S cell line. Analysis of the N-glycan in rhodopsin expressed by the HEK293S GnTI− stable cell line showed it to be Man5GlcNAc2. In a larger-scale expression experiment (1.1 liter) a WT opsin production level of 6 mg/liter was obtained. Further, the toxic constitutively active rhodopsin mutant, E113Q/E134Q/M257Y, previously shown to require inducible expression, has now been expressed in an HEK293S GNTI−-inducible cell line at levels comparable with those obtained with WT rhodopsin.

https://www.pnas.org/content/pnas/99/21/13419.full.pdf

Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line

Immortalized human chondrocytes were established by transfection of primary cultures of juvenile costal chondrocytes with vectors encoding simian virus 40 large T antigen and selection in suspension culture over agarose. Stable cell lines were generated that exhibited chondrocyte morphology, continuous proliferative capacity (> 80 passages) in monolayer culture in serum-containing medium, and expression of mRNAs encoding chondrocyte-specific collagens II, IX, and XI and proteoglycans in an insulin-containing serum substitute. They did not express type X collagen or versican mRNA. These cells synthesized and secreted extracellular matrix molecules that were reactive with monoclonal antibodies against type II collagen, large proteoglycan (PG-H, aggrecan), and chondroitin-4- and chondroitin-6-sulfate. Interleukin-1 beta (IL-1 beta) decreased the levels of type II collagen mRNA and increased the levels of mRNAs for collagenase, stromelysin, and immediate early genes (egr-1, c-fos, c-jun, and jun-B). These cell lines also expressed reporter gene constructs containing regulatory sequences (-577/+3,428 bp) of the type II collagen gene (COL2A1) in transient transfection experiments, and IL-1 beta suppressed this expression by 50-80%. These results show that immortalized human chondrocytes displaying cartilage-specific modulation by IL-1 beta can be used as a model for studying normal and pathological repair mechanisms.

/pdf/interleukin-1-beta-modulated-gene-expression-in-immortalized-58zl9rfiry.pdf

Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes.

N-Glycan branching in the medial-Golgi generates ligands for lattice-forming lectins (e.g., galectins) that regulate surface levels of glycoproteins including epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) receptors. Moreover, functional classes of glycoproteins differ in N-glycan multiplicities (number of N-glycans/peptide), a genetically encoded feature of glycoproteins that interacts with metabolic flux (UDP-GlcNAc) and N-glycan branching to differentially regulate surface levels. Oncogenesis increases beta1,6-N-acetylglucosaminyltransferase V (encoded by Mgat5) expression, and its high-affinity galectin ligands promote surface retention of growth receptors with a reduced dependence on UDP-GlcNAc. Mgat5(-/-) tumor cells are less metastatic in vivo and less responsive to cytokines in vitro, but undergo secondary changes that support tumor cell proliferation. These include loss of Caveolin-1, a negative regulator of EGF signaling, and increased reactive oxygen species, an inhibitor of phosphotyrosine phosphatases. These studies suggest a systems approach to cancer treatment where the surface distribution of receptors is targeted through metabolism and N-glycan branching to induce growth arrest.

/pdf/n-glycans-in-cancer-progression-5fc7ey5cy8.pdf

N-Glycans in cancer progression

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an a-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.

/pdf/comparison-of-biological-activity-among-nonfucosylated-5c55wusob5.pdf

Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types.

High-frequency transfection of CHO cells has been achieved for several plasmids, a cosmid library, and genomic DNA using Polybrene and dimethyl sulfoxide. All plasmid transfectants examined were stable and exhibited plasmid sequences in genomic DNA. The method is simple, reproducible, and succeeded with several independent CHO clones in the presence or the absence of carrier DNA, even at very low concentrations of plasmid DNA.

High-frequency transfection of CHO cells using polybrene.

The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase alpha.

Two CHO glycosylation mutants that were previously shown to lack N-linked carbohydrates with GlcNAc beta 1,6Man alpha 1,6 branches, and to belong to the same genetic complementation group, are shown here to differ in the activity of N-acetylglucosaminyltransferase V (GlcNAc-TV) (UDP-GlcNA: alpha 1,6mannose beta-N-acetylglucosaminyltransferase V). One mutant, Lec4, has no detectable GlcNAc-TV activity whereas the other, now termed Lec4A, has activity equivalent to that of parental CHO in detergent cell extracts. However, Lec4A GlcNAc-TV can be distinguished from CHO GlcNAc-TV on the basis of its increased sensitivity to heat inactivation and its altered subcellular compartmentalization. Sucrose density gradient fractionation shows that the major portion of GlcNAc-TV from Lec4A cells cofractionates with membranes of the ER instead of Golgi membranes where GlcNAc-TV is localized in parental CHO cells. Other experiments show that Lec4A GlcNAc-TV is not concentrated in lysosomes, or in a post-Golgi compartment, or at the cell surface. The altered localization in Lec4A cells is specific for GlcNAc-TV because two other Lec4A Golgi transferases cofractionate at the density of Golgi membranes. The combined data suggest that both lec4 and lec4A mutations affect the structural gene for GlcNAc-TV, causing either the loss of GlcNAc-TV activity (lec4) or its miscompartmentalization (lec4A). The identification of the Lec4A defect indicates that appropriate screening of different glycosylation-defective mutants should enable the isolation of other mammalian cell trafficking mutants.

/pdf/the-lec4a-cho-glycosylation-mutant-arises-from-2k4nkz5h8w.pdf

The Lec4A CHO glycosylation mutant arises from miscompartmentalization of a Golgi glycosyltransferase.

Lec1A Chinese hamster ovary cell mutants appear to arise from a structural alteration in N-acetylglucosaminyltransferase I

Lec1 CHO cell glycosylation mutants are defective in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and therefore cannot convert the oligomannosyl intermediate (Man5GlcNAc2Asn) into complex carbohydrates. Lec1A CHO cell mutants have been shown to belong to the same genetic complementation group but exhibit different phenotypic properties. Evidence is presented that lec1A represents a new mutation at the lec1 locus resulting in partial loss of GlcNAc-TI activity. Structural studies of the carbohydrates associated with vesicular stomatitis virus grown in Lec1A cells (Lec1A/VSV) revealed the presence of biantennary and branched complex carbohydrates as well as the processing intermediate Man5GlcNAc2Asn. By contrast, the glycopeptides from virus grown in CHO cells (CHO/VSV) possessed only fully processed complex carbohydrates, whereas those from Lec1/VSV were almost solely of the Man5GlcNAc2Asn intermediate type. Therefore, the Lec1A glycosylation phenotype appears to result from the partial processing of N-linked carbohydrates because of reduced GlcNAc-TI action on membrane glycoproteins. Genetic experiments provided evidence that lec1A is a single mutation affecting GlcNAc-TI activity. Lec1A mutants could be isolated at frequencies of 10(-5) to 10(-6) from unmutagenized CHO cell populations by single-step selection, a rate inconsistent with two mutations. In addition, segregants selected from Lec1A X parental cell hybrid populations expressed only Lec1A or related lectin-resistant phenotypes and did not include any with a Lec1 phenotype. The Lec1A mutant should be of interest for studies on the mechanisms that control carbohydrate processing in animal cells and the effects of reduced GlcNAc-TI activity on the glycosylation, translocation, and compartmentalization of cellular glycoproteins.

William G. Chaney

Papers

High-frequency transfection of CHO cells using polybrene.

The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase alpha.

The Lec4A CHO glycosylation mutant arises from miscompartmentalization of a Golgi glycosyltransferase.

Lec1A Chinese hamster ovary cell mutants appear to arise from a structural alteration in N-acetylglucosaminyltransferase I

Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity.