Differences In N-linked Glycosylation Research Articles

The epidermal growth factor ortholog of ectromelia virus activates EGFR/ErbB1 and demonstrates mitogenic function in vitro

Many poxviruses produce proteins that are related to epidermal growth factor (EGF). Prior genome sequencing of ectromelia virus revealed a gene predicted to produce a protein with homology to EGF, which we refer to as ectromelia growth factor (ECGF). ECGF is truncated relative to vaccinia growth factor (VGF) because the former lacks a transmembrane domain. We show these proteins can experience differential N-linked glycosylation. Despite these differences, both proteins maintain the six conserved cysteine residues important for the function of EGF. Since ECGF has not been characterized, our objective was to determine if it can act as a growth factor. We added ECGF to cultured cells and found that the EGF receptor becomes activated, S-phase was induced, doubling time decreased, and in vitro wound healing occurred faster compared to untreated cells. In summary, we demonstrate that ECGF can act as a mitogen in a similar manner as VGF.

Abstract PO-094: Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors

Abstract Prostate cancer is the most commonly diagnosed cancer in men worldwide. A critical knowledge gap in prostate cancer biology is the molecular events underlining higher incidence and mortality rate in Black men. Identifying molecular features that separate racial disparities is a critical step in prostate cancer research that could lead to predictive biomarkers and personalized therapy. N-linked glycosylation is a required co-translational event during protein folding that modulates many biological processes, such as cell adhesion, immune modulation, cell-matrix interactions, and cell proliferation. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a new and innovative technique that combines mass spectrometry with imaging, enabling the detection of glycans with spatial distribution. Herein, we performed MALDI-MSI analysis to characterize the N-glycan profile from tissue microarrays of over 100 patient tumors banked at the University of Kentucky with over 10 years of follow-up data. Additionally, we performed MALDI-MSI analysis on large sections of prostate cancer to define the regional distribution of N-glycans within the tumor microenvironment. We successfully identified 46 unique glycans from readily available formalin-fixed paraffin-embedded prostate tissue, and found significant N- glycan dysregulation between benign and prostate tumor tissue across all patient groups. High mannose as well as tri- and tetra-antennary N-glycans were predominantly found in tumor tissue and correlate with increased tumor grade. Surprisingly, several species of N-glycans were profoundly different between early grade prostate tumors resected from White and Black patients. Further, these glycans predict opposing overall survival between White and Black patients with prostate cancer. These data suggest differential N-linked glycosylation underline the racial disparity of prostate cancer prognosis. Our study highlights the potential clinical applications of MALDI-MSI for digital pathology and biomarker applications, and reveals molecular features that contribute to the racial disparity in diagnosis and survival of prostate cancer patients. Citation Format: Lindsey R. Conroy, Lyndsay E.A. Young, Grant L. Austin, Alexanda E. Stanback, Derek B Allison, Matthew S. Gentry, Richard R. Drake, Ramon C. Sun. Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-094.

Abstract PO-085: Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors

Abstract Prostate cancer is the most commonly diagnosed cancer in men worldwide. A critical knowledge gap in prostate cancer biology is the molecular events underlining higher incidence and mortality rate in Black men. Identifying molecular features that separate racial disparities is a critical step in prostate cancer research that could lead to predictive biomarkers and personalized therapy. N-linked glycosylation is a required co-translational event during protein folding that modulates many biological processes, such as cell adhesion, immune modulation, cell-matrix interactions, and cell proliferation. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a new and innovative technique that combines mass spectrometry with imaging, enabling the detection of glycans with spatial distribution. Herein, we performed MALDI-MSI analysis to characterize the N-glycan profile from tissue microarrays of over 100 patient tumors banked at the University of Kentucky with over 10 years of follow-up data. Additionally, we performed MALDI-MSI analysis on large sections of prostate cancer to define the regional distribution of N-glycans within the tumor microenvironment. We successfully identified 46 unique glycans from readily available formalin-fixed paraffin-embedded prostate tissue, and found significant N-glycan dysregulation between benign and prostate tumor tissue across all patient groups. High mannose as well as tri- and tetra-antennary N-glycans were predominantly found in tumor tissue and correlate with increased tumor grade. Surprisingly, several species of N-glycans were profoundly different between early grade prostate tumors resected from White and Black patients. Further, these glycans predict opposing overall survival between White and Black patients with prostate cancer. These data suggest differential N-linked glycosylation underline the racial disparity of prostate cancer prognosis. Our study highlights the potential clinical applications of MALDI-MSI for digital pathology and biomarker applications, and reveals molecular features that contribute to the racial disparity in diagnosis and survival of prostate cancer patients. Citation Format: Lindsey R. Conroy, Lyndsay E.A. Young, Grant L. Austin, Alexandra E. Stanback, Derek B. Allison, Matthew S. Gentry, Richard R. Drake, Ramon C. Sun. Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-085.

Addiction associated N40D mu-opioid receptor variant modulates synaptic function in human neurons.

The OPRM1 A118G single nucleotide polymorphism (SNP rs1799971) gene variant encoding the N40D ¼-opioid receptor (MOR) has been associated with dependence on opiates and other drugs of abuse but its mechanism is unknown. The frequency of G-allele carriers is ~40% in Asians, ~16% in Europeans, and ~3% in African-Americans. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy. Here we generated homozygous N40D subject-specific induced inhibitory neuronal cells (iNs) from seven human induced pluripotent stem (iPS) cell lines from subjects of European descent (both male and female) and probed the impact of N40D MOR regulation on synaptic transmission. We found that D40 iNs exhibit consistently stronger suppression (versus N40) of spontaneous inhibitory postsynaptic currents (sIPSCs) across multiple subjects. To mitigate the confounding effects of background genetic variation on neuronal function, the regulatory effects of MORs on synaptic transmission were recapitulated in two sets of independently engineered isogenic N40D iNs. In addition, we employed biochemical analysis and observed differential N-linked glycosylation of human MOR N40D. This study identifies neurophysiological and molecular differences between human MOR variants that may predict altered opioid responsivity and/or dependence in this subset of individuals.

Generation and Analysis of Novel Plant-Derived Antibody-Based Therapeutic Molecules against West Nile Virus

Previously, our group engineered a plant-derived monoclonal antibody (MAb) (pHu-E16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed several pHu-E16 variants to improve its efficacy. These variants included a single-chain variable fragment (scFv) of pHu-E16 fused to the heavy chain (HC) constant domains (CH 1-3) of human IgG (pHu-E16scFv-CH 1-3) and a tetravalent molecule (Tetra pHu-E16) assembled from pHu-E16scFv-CH 1-3 with a second pHu-E16scFv fused to the light chain (LC) constant region. pHu-E16scFv-CH 1-3 and Tetra pHu-E16 were efficiently expressed and assembled in plants. To assess the impact of differences in N-linked glycosylation on pHu-E16 variant assembly and function, we expressed additional pHu-E16 variants with various combinations of HC and LC components. Our study revealed that proper pairing of HC and LC was essential for the complete N-glycan processing of antibodies in both plant and animal cells. Associated with their distinct N-glycoforms, pHu-E16, pHu-E16scFv-CH 1-3 and Tetra pHu-E16 exhibited differential binding to C1q and specific Fcγ receptors (FcγR). Notably, none of the plant-derived Hu-E16 variants showed antibody-dependent enhancement (ADE) activity in CD32A+ human cells, suggesting the potential of plant-produced antibodies to minimize the adverse effect of ADE. Importantly, all plant-derived MAb variants exhibited at least equivalent in vitro neutralization and in vivo protection in mice compared to mammalian cell-produced Hu-E16. This study demonstrates the capacity of plants to express and assemble a large, complex and functional IgG-like tetravalent mAb variant and also provides insight into the relationship between MAb N-glycosylation, FcγR and C1q binding, and ADE. These new insights may allow the development of safer and cost effective MAb-based therapeutics for flaviviruses, and possibly other pathogens.

Tissue- and development-specific glycosylation states of the natriuretic peptide receptors guanylyl cyclase-A (GC-A) and GC-B

Background The natriuretic peptides (NPs) atrial NP (ANP), B-type NP (BNP) and C-type NP (CNP) elicit cellular effects by binding to transmembrane proteins with guanylyl cyclase (GC) activity, referred to as GC-A (representing the common receptor for ANP and BNP) and GC-B (the CNP receptor). GC-A and GC-B are structurally closely related proteins with very similar cDNA-deduced molecular masses in the range of 115 kDa, but their native molecular weights are significantly (by up to 20 kDa) higher due to glycosylation. Differential glycosylation of cell surface receptors can affect folding, trafficking, ligand binding and/or agonistinduced signal transduction. Moreover, specific temporal expression patterns of carbohydrate structures are frequently involved in developmental processes. Hitherto, however, the glycosylation states of GC-A and GC-B in vivo are largely unknown, since currently available data are based to a great extent on studies with receptor-transfected cell lines. This investigation aimed to characterize the sizes and posttranslational modifications of native GC-A and GCB in different tissues of adult rats and mice. Based on increasing evidence for specific activities of NPs and their receptors in the developing brain (1-3), we examined the glycosylation of GC-A and GC-B during postnatal brain development. Methods and results Western blot (as well as receptor/ligand crosslinking) experiments and treatments with carbohydrate-digesting enzymes served to investigate the sizes and posttranslational modifications of the two receptors. Due to marked tissue-specific differences in N-linked glycosylation, mean apparent molecular masses of both rat and mouse GC-A range from 119 kDa in brain to values between 124 (lung) and 132 (heart) kDa in peripheral tissues. The reduced glycosylation in brain seems to be central nervous system (CNS)-specific, since the pituitary, which contains both CNS and CNS-unrelated tissue, co-expresses the CNS-typical (119 kDa, reference: olfactory bulb) and the peripheral (up to 130 Da) forms of GC-A. GC-B sizes differed in the same manner between peripheral organs, indicating equal tissue-dependent influences on oligosaccharide processing at GC-A and GC-B. A higher (by 3 kDa) total amount of N-linked carbohydrates suggests usage of an additional consensus N-glycosylation site (7 instead of 6 in the case of GC-A). The existence of region-specific GC-B size variants in cerebellum and olfactory bulb discriminates this receptor from GC-A in the adult brain. Both GC-A and GC-B (but not two other glycosylated membrane proteins examined) are hyperglycosylated at N-linked sites during early postnatal brain development. At postnatal day 1, the vast majority of GC-B (but not GC-A) contain additionally an O-linked carbohydrate modification.

Ross River Virus Envelope Glycans Contribute to Type I Interferon Production in Myeloid Dendritic Cells

Alphaviruses are mosquito-transmitted viruses that cause significant human disease, and understanding how these pathogens successfully transition from the mosquito vector to the vertebrate host is an important area of research. Previous studies demonstrated that mosquito and mammalian-cell-derived alphaviruses differentially induce type I interferons (alpha/beta interferon [IFN-alpha/beta]) in myeloid dendritic cells (mDCs), where the mosquito cell-derived virus is a poor inducer of IFN-alpha/beta compared to the mammalian-cell-derived virus. Furthermore, the reduced IFN-alpha/beta induction by the mosquito cell-derived virus is attributed to differential N-linked glycosylation. To further evaluate the role of viral envelope glycans in regulating the IFN-alpha/beta response, studies were performed to assess whether the mosquito cell-derived virus actively inhibits IFN-alpha/beta induction or is simply a poor inducer of IFN-alpha/beta. Coinfection studies using mammalian- and mosquito cell-derived Ross River virus (mam-RRV and mos-RRV, respectively) indicated that mos-RRV was unable to suppress IFN-alpha/beta induction by mam-RRV in mDC cultures. Additionally, a panel of mutant viruses lacking either individual or multiple N-linked glycosylation sites was used to demonstrate that N-linked glycans were essential for high-level IFN-alpha/beta induction by the mammalian-cell-derived virus. These results suggest that the failure of the mosquito cell-derived virus to induce IFN-alpha/beta is due to a lack of complex carbohydrates on the virion rather than the active suppression of the DC antiviral response.

A novel platform for biologically active recombinant human interleukin‐13 production

Interleukin-13 (IL-13) is a pleiotropic regulatory cytokine with the potential for treating several human diseases, including type-1 diabetes. Thus far, conventional expression systems for recombinant IL-13 production have proven difficult and are limited by efficiency. In this study, transgenic plants were used as a novel expression platform for the production of human IL-13 (hIL-13). DNA constructs containing hIL-13 cDNA were introduced into tobacco plants. Transcriptional expression of the hIL-13 gene in transgenic plants was confirmed by reverse transcriptase-polymerase chain reaction and Northern blotting. Western blot analysis showed that the hIL-13 protein was efficiently accumulated in transgenic plants and present in multiple molecular forms, with an expression level as high as 0.15% of total soluble protein in leaves. The multiple forms of plant-derived recombinant hIL-13 (rhIL-13) are a result of differential N-linked glycosylation, as revealed by enzymatic and chemical deglycosylation, but not of disulphide-linked oligomerization. In vitro trypsin digestion indicated that plant rhIL-13 was more resistant than unglycosylated control rhIL-13 to proteolysis. The stability of plant rhIL-13 to digestion was further supported with simulated gastric and intestinal fluid digestion. In vitro bioassays using a factor-dependent human erythroleukaemic cell line (TF-1 cells) showed that plant rhIL-13 retained the biological functions of the authentic hIL-13 protein. These results demonstrate that transgenic plants are superior to conventional cell-based expression systems for the production of rhIL-13. Moreover, transgenic plants synthesizing high levels of rhIL-13 may prove to be an attractive delivery system for direct oral administration of IL-13 in the treatment of clinical diseases such as type-1 diabetes.

Protein Kinase A Affects Trafficking of the Vesicular Monoamine Transporters in PC12 Cells

Previous studies have shown that the vesicular monoamine transporter 2 (VMAT2) is localized to both large dense core vesicles and synaptic vesicles in vivo. However, when exogenously expressed in PC12 cells, VMAT2 localizes only to large dense core vesicles. This distribution is similar to that of the endogenous vesicular monoamine transporter 1 (VMAT1) in PC12 cells. When VMAT2 was expressed in a protein kinase A (PKA)-deficient PC12 cell line it localized to synaptic-like microvesicles. Expression of recombinant VMAT1 in the same cell line showed a heterogeneous distribution to both large dense core vesicles and synaptic-like microvesicles. Coexpression of the PKA catalytic subunit partially restored trafficking of both VMAT2 and VMAT1 to large dense core vesicles; treatment of wild-type PC12 cells with the PKA inhibitor H89 increased VMAT2 on synaptic-like microvesicles. The VMAT1 and VMAT2 in large dense core vesicles exhibit a larger molecular size than those located on synaptic-like microvesicles. This difference is due to differential N-linked glycosylation. In vitro phosphorylation experiments show that PKA does not phosphorylate VMAT2. A chimera containing the VMAT2 cytoplasmic C-terminus fused to vesicular acetylcholine transporter (VAChT) shows mislocalization to synaptic-like microvesicles and VAChT-like glycosylation in the PKA-deficient cell line. However, coexpression with PKA changes the chimera's trafficking to large dense core vesicles and increases the molecular size. These results suggest that protein kinase A affects the formation and/or composition of VMAT trafficking complexes.

Differential N-linked glycosylation of human immunodeficiency virus and Ebola virus envelope glycoproteins modulates interactions with DC-SIGN and DC-SIGNR.

The C-type lectins DC-SIGN and DC-SIGNR [collectively referred to as DC-SIGN(R)] bind and transmit human immunodeficiency virus (HIV) and simian immunodeficiency virus to T cells via the viral envelope glycoprotein (Env). Other viruses containing heavily glycosylated glycoproteins (GPs) fail to interact with DC-SIGN(R), suggesting some degree of specificity in this interaction. We show here that DC-SIGN(R) selectively interact with HIV Env and Ebola virus GPs containing more high-mannose than complex carbohydrate structures. Modulation of N-glycans on Env or GP through production of viruses in different primary cells or in the presence of the mannosidase I inhibitor deoxymannojirimycin dramatically affected DC-SIGN(R) infectivity enhancement. Further, murine leukemia virus, which typically does not interact efficiently with DC-SIGN(R), could do so when produced in the presence of deoxymannojirimycin. We predict that other viruses containing GPs with a large proportion of high-mannose N-glycans will efficiently interact with DC-SIGN(R), whereas those with solely complex N-glycans will not. Thus, the virus-producing cell type is an important factor in dictating both N-glycan status and virus interactions with DC-SIGN(R), which may impact virus tropism and transmissibility in vivo.

Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease.

Human surfactant protein-B (SP-B), a hydrophobic protein, is essential for normal lung function. SP-B is expressed and secreted by specific lung cell types, i.e. alveolar type II and Clara cells, of the respiratory epithelium. The SP-B precursor (42 kDa) undergoes post-translational processing to generate an 8 kDa mature SP-B. A single-nucleotide polymorphism (SNP) at nucleotide 1580 (C/T) in exon 4 of SP-B that changes amino acid 131 from threonine to isoleucine (Thr131-->Ile) is associated with several pulmonary diseases. The Thr131-->Ile substitution can eliminate a potential N-linked glycosylation site, Asn129-Gln-Thr131, which is present in the SP-B variant of the C allele (ACT/Thr) but not in that of the T allele (ATT/Ile). To determine whether the C allele SP-B variant is indeed glycosylated at Asn(129)-Gln-Thr131, we first generated stably transfected Chinese hamster ovary cell lines that expressed each version of SP-B, and developed specific SP-B polyclonal anti-peptide antibodies. Using both the stably transfected cell lines and fetal lung explants, we observed that the C allele variant is indeed glycosylated at the Asn129-Gln-Thr131 site, whereas the T allele variant, which served as a control, is not. In addition, we also confirmed that both SP-B variants contain another N-linked glycosylation site, Asn311-Ser-Ser313. Given its association with several pulmonary diseases, this finding provides useful information for future studies in disease systems associated with this SNP. Further, we speculate that, given the fact that this SNP is found frequently in the general population, N-linked glycosylation at residue Asn129 interferes with SP-B processing, secretion and folding under certain disease conditions.

Large scale production of recombinant human lactoferrin in the milk of transgenic cows.

The limited capacity of current bioreactors has led the biopharmaceutical industry to investigate alternative protein expression systems. The milk of transgenic cattle may provide an attractive vehicle for large-scale production of biopharmaceuticals, but there have been no reports on the characteristics of such recombinant proteins. Here we describe the production of recombinant human lactoferrin (rhLF), an iron-binding glycoprotein involved in innate host defense, at gram per liter concentrations in bovine milk. Natural hLF from human milk and rhLF had identical iron-binding and -release properties. Although natural hLF and rhLF underwent differential N-linked glycosylation, they were equally effective in three different in vivo infection models employing immunocompetent and leukocytopenic mice, and showed similar localization at sites of infection. Taken together, the results illustrate the potential of transgenic cattle in the large-scale production of biopharmaceuticals.

Central nervous system-specific glycosylation of the type A natriuretic peptide receptor.

Physiological effects of atrial natriuretic peptide (ANP) are thought to be mediated by binding to and activation of a widely expressed membrane receptor, termed guanylyl cyclase (GC)-A. During comparative analyses of ANP receptor expression in various rat and bovine tissues, by UV light-induced affinity cross-linking to (125)I-ANP, we uncovered a size heterogeneity of GC-A, detectable as a 130-kDa protein in peripheral and as a 122-kDa protein in central nervous system tissues. This heterogeneity could be explained by differences in N-linked glycosylation, because treatments with N-glycosidase F reduced the apparent molecular weights of both receptor variants to the same value of 116,000. As judged by displacement experiments, the two receptor glycoforms did not differ in their binding affinities for natriuretic peptides. Assessments of GC activities did not reveal any difference in ANP-induced generation of the second messenger, cyclic GMP. The examination of GC-A expression in brain during ontogeny revealed an alteration of the apparent molecular mass during early postnatal development from the 130-kDa to the 122-kDa form, suggesting a change in oligosaccharide processing. This study provides a reliable method for characterizing GC-A expression and identifies, for the first time, a differential glycosylation of this receptor in vivo.

Characterization of glycosylated Gag expressed by a neurovirulent murine leukemia virus: identification of differences in processing in vitro and in vivo.

The neuroinvasiveness of a chimeric murine retrovirus, CasFrKP (KP), is dependent on the expression of glycosylated Gag (gp85gag). This viral protein is the product of alternate translation initiation 88 codons upstream of and in frame with the initiation codon of pr65gag, the precursor of the viral core proteins. Although expression of glycosylated Gag affects virus spread in the spleen, it appears not to affect virus spread in vitro in fibroblast cell lines (J. L. Portis et al., J. Virol. 68:3879-3887, 1994). The differential effects of this protein in vitro and in vivo have not been explained, and its function is unknown. We have here compared the in vitro processing of this molecule with that expressed in spleens of infected mice. In vitro, gp85gag was cleaved near the middle of the molecule, releasing the C-terminal half (containing capsid and nucleocapsid domains of pr65gag) as a secreted glycoprotein. The N-terminal half of the protein was associated with the plasma membrane as a approximately 55-kDa glycoprotein bearing the matrix domain of pr65gag as well as the N-terminal 88 residue L domain. This processing scheme was also observed in vivo, although two differences were seen. There were differences in N-linked glycosylation of the secreted form of the protein expressed in the spleen. In addition, whereas the membrane-associated species assumed the orientation of a type II integral membrane protein (N(cyto) C(exo)) in fibroblasts in vitro, a subpopulation of spleen cells was detected in which the N terminus of the protein was exposed at the cell surface. These results suggest that the differential effects of glycosylated Gag expression in vivo and in vitro may be related to differences in posttranslational processing of the protein.

The Myeloid Differentiation Antigen CD14 is N‐ and O‐Glycosylated

The myeloid differentiation antigen CD14 acts as the major receptor for bacterial lipopolysaccharide (LPS). A soluble form of the protein (sCD14) is present in human serum which functions as a soluble LPS receptor. We have compared the isoform patterns of soluble CD14 derived from human serum and of the recombinant proteins produced by CHO cells transfected with either the wild-type CD14 gene or with a cDNA coding for a truncated protein which lacks the C-terminal 21 amino acids [sCD14-(1-335)-peptide]. Using SDS/PAGE, two dominant isoforms (53 and 50 kDa) and two minor forms (46 and 43 kDa) can be detected in serum as well as in the supernatants of both transfectants. sCD14 is a glycoprotein which carries N- and O-linked carbohydrates. The different isoforms of sCD14-(1-335)-peptide are due to differences in the content of N-linked sugars. However after the removal of N- and O-linked carbohydrates from serum- and CHO-derived wild-type proteins, two isoforms are still present. These results indicate that N-linked glycosylation contributes to but does not fully explain the different forms of soluble CD14. We further examined whether the mutation of individual N-linked glycosylation sites influences the expression of membrane-bound and soluble CD14 forms and the ability of the membrane-bound molecule to bind LPS. As with the wild-type proteins, the different isoforms of the soluble mutants are partially due to differences in N-linked glycosylation. A truncated mutant which lacks the two N-terminal glycosylation sites {[Asp18, Asp132]CD14-(1-335)peptide} does not give rise to multiple forms on SDS gels. Like CD14-(1-335)-peptide, this mutant is not expressed on the cell surface suggesting that a smaller isoform present in the wild-type preparations results from proteolytic cleavage of the membrane-bound molecule. N-linked carbohydrates do not seem to be important for the binding of LPS to membrane-bound CD14.

Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation.

CD44 is a major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA). However, the ability of CD44 to bind ligand is strictly regulated. Three activation states of CD44 have been demonstrated: (a) inactive; (b) inducible (by certain CD44-specific mAb); and (c) constitutively active. Starting with two parental cell lines expressing CD44 in the inactive state, a pre-B cell (RAW 253) and a fibroblast (L cells), we used fluorescence-activated cell sorting with fluorescein-conjugated hyaluronan in the presence of inducing mAb to derive variant cell lines with CD44 in the inducible state. Constitutively active derivatives were isolated from the inducible variants by a further round of fluorescence-activated cell sorting in the absence of inducing antibody. However, constitutively active variants could not be isolated directly from parental cells expressing CD44 in the inactive state. These results suggest that two genetic events must occur to obtain an active CD44-HA receptor from an inactive receptor. Variant and parental cell-derived CD44 molecules exhibited differences in migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were partly attributable to differences in N-linked glycosylation. Furthermore, culture in tunicamycin for 2-3 d converted parental and inducible cell lines into cells showing constitutive CD44-mediated HA binding. Also, removal of cell surface glycosaminoglycan chains by culture of cells in p-nitrophenyl beta-D-xylopyranoside or treatment with chondroitinase ABC resulted in conversion of cells with an inactive CD44 receptor to an inducible state. These results indicate that carbohydrate side chains of CD44 and/or other molecules on the cell surface that interact with CD44 are potentially involved in regulating the HA-binding function of CD44 on the cell surface.

Heterogenous glycosylation of ICAM‐3 and lack of interaction with Mac‐1 and p150,95

Intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAM-3 have been identified as counter-receptors for the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). The other leukocyte integrins, Mac-1 and p150,95, also interact with ICAM-1. ICAM-1 and ICAM-3 are highly homologous, and an undefined ligand for Mac-1 is present on neutrophils where ICAM-3 is well expressed. In addition, glycosylation has been shown to affect the interaction of ICAM-1 with Mac-1. We therefore sought to characterize ICAM-3 heterogeneity and determine whether ICAM-3 was a ligand for either Mac-1 or p150,95. Despite extensive differences in N-linked glycosylation, ICAM-3 purified from lymphoid cells and from neutrophils supports adhesion of LFA-1-bearing cells equally well; however, neither supports adhesion of Mac-1 or p150,95-expressing chinese hamster ovary cell transfectants. Similarly, purified Mac-1 does not support adhesion of ICAM-2 or ICAM-3-expressing L cell transfectants. ICAM-3 on neutrophils does not participate in Mac-1-dependent homotypic aggregation. Thus, ICAM-3 is not a counter-receptor for either Mac-1 or p150,95.

Developmentally Regulated Expression of a Novel 59-kDa Product of the Major Surface Protease (Msp or gp63) Gene Family of Leishmania Chagasi

All species of Leishmania express a major surface protease (Msp or gp63) that facilitates the interactions of the parasite with its environment at several steps in its life cycle. The msp gene family in Leishmania chagasi contains three classes of genes whose mRNAs are differentially expressed during parasite growth. Logarithmic phase (low infectivity) promastigotes express only 63-kDa versions of Msp, whereas stationary phase (high infectivity) promastigotes express both 63- and 59-kDa Msps. The different migrations of the 59- and 63-kDa proteins on acrylamide gels are not due to differences in N-linked glycosylation or the membrane anchor. Plasmid transfections of Leishmania demonstrate that mspS2 of the stationary gene class encodes a 59-kDa protein. Expression of the 59-kDa protein in stationary phase promastigotes ceases after about 12 weeks of in vitro cultivation when the parasites become attenuated. Attenuated parasites can be stimulated to re-express the 59-kDa Msp by passage through mice followed by several in vitro passages of recovered promastigotes. Amastigotes express yet another subset of Msp proteins. Thus, the 59-kDa product of mspS2 is expressed only in stationary phase promastigotes and only after recent exposure to environmental changes encountered in the mammalian host cell.

Expression of an endogenous asialoglycoprotein receptor in a human intestinal epithelial cell line, Caco-2

We have previously shown that rat asialoglycoprotein receptor expressed in the intestine and liver differ in mRNA size, cell surface distribution, and ratio of compositional protein subunits. In this study, we examined a well characterized intestinal epithelial cell line, Caco-2, as a potential model for studying endogenous receptor in a polarized cell line. Both subunits H1 and H2 of human asialoglycoprotein receptor were detected in Caco-2 cells by Western blots using subunit-specific antisera raised against the hepatic receptor. Antigenic receptor level in fully differentiated Caco-2 cells was approx. 1 3 to 1 2 the level of hepatic HepG2 cells and H1 was the dominant subunit in both cell lines. The apparent size of H1 and H2 in Caco-2 cells was not the same as that in HepG2 cells, due to differences in N-linked glycosylation. Consistent with this finding, Northern blot analysis showed that receptor mRNA in the two cell types was of identical size. In pulse-chase experiments H1 was first detected as a ‘high-mannose’ precursor (40 kDa) in Caco-2 cells that was converted to mature H1 (43 kDa) with a half-life of approx. 60 min. Antigenic levels of H1 and H2 in undifferentiated Caco-2 cells were low, but increased rapidly during cell differentiation, reaching a peak level at 7 days after confluence. Immunocytochemical staining and domain-selective cell surface biotinylation assays showed that the ASGP-R was predominantly localized in the basolateral domain. The receptor in Caco-2 cells was capable of mediating specific uptake and degradation of [ 125I]asialoorosomucoid. The ligand uptake capacity of the basolateral surface of was approx. 10-fold higher than the apical. These characteristics (H1 subunit and basolateral predominance) of the receptor in Caco-2 cells, resembles the hepatic receptor. We conclude that Caco-2 cells endogenously express an ectopic hepatic-type functional asialoglycoprotein receptor.

Differential glycosylation of the GLUT1 glucose transporter in brain capillaries and choroid plexus

The sodium-independent GLUT1 glucose transporter is expressed in high density in human erythrocytes and in tissues which serve a barrier function. In the polarized endothelial cells of the brain capillaries, which comprise the blood-brain barrier (BBB), GLUT1 is expressed on both apical and basolateral membranes; however, in the epithelium of the choroid plexus, GLUT1 expression is restricted to the basolateral surface. The present study examined whether these differences in subcellular localization of GLUT1 at the BBB and choroid plexus could be correlated with differential N-linked or O-linked glycosylation of the protein. Western blot analysis of solubilized brain capillaries (BC) and choroid plexus (CP) revealed that while the BC GLUT1 had an average molecular mass identical to that of the purified human erythrocyte transporter (54 kDa), the CP GLUT1 was of lower molecular mass (47 kDa). Treatment of brain capillaries and choroid plexus with N-glycanase resulted in a shift in the mobility of the GLUT1 of both samples to a lower molecular mass of 42 kDa; however, in contrast, treatment with O-glycanase produced no change in the mobility patterns of GLUT1, but did result in O-linked deglycosylation of another BBB marker, γ-glutamyl transpeptidase. In conclusion, BBB and choroid plexus GLUT1 are subject to differential N-linked glycosylation with the protein having an N-linked carbohydrate side chain of higher molecular mass at the BBB in comparison to the choroid plexus.