Affinity chromatography of branched oligosaccharides in rat liver beta-glucuronidase.

Rat liver microsomal and lysosomal beta-glucuronidase-derived glycopeptides were obtained by extensive Pronase digestion followed by N-[14C]acetylation and desialylation by neuraminidase treatment. These glycopeptides were studied by sequential chromatography on lectin-affinity columns such as concanavalin A, lentil lectin, Phaseolus vulgaris erythroagglutinin, Ricinus communis agglutinin I, Triticum vulgaris agglutinin, Glycine max agglutinin and Ulex europaeus agglutinin. Using serial lectin affinity chromatography approach combined with neuraminidase treatment allowed us to show the unexpected presence of complex tri- and/or tetraantennary type glycans (40.8 and 17.0% for microsomal and lysosomal enzyme, respectively). Moreover, the application of neuraminidase treatment revealed that complex biantennary type glycans, present on lysosomal beta-glucuronidase, are almost fully sialylated while the same type of glycans present on microsomal enzyme do not contain sialic acid. Furthermore, the results obtained confirmed that microsomal and lysosomal beta-glucuronidases possess high mannose and/or hybrid type glycans (19.6 and 36.6%, respectively), and complex biantennary type glycans (38.9 and 46.4%, respectively).

Study of the sugar chains of recombinant human amyloid precursor protein produced by Chinese hamster ovary cells.

The N- and O-glycans of recombinant amyloid precursor protein (APP), purified from Chinese hamster ovary cells transfected with the human 695-amino acid form of APP, were separately released by hydrazinolysis under different conditions. The reducing ends of the released N- and O-glycans were reduced with NaB3H4 and derivatized with 2-aminobenzamide (2AB), respectively. After acidic N-glycans were obtained by anion-exchange column chromatography, these were converted to neutral oligosaccharides by sialidase digestion, demonstrating that their acidic nature was entirely due to sialylation. The sialidase-treated N-glycans were then fractionated by lectin column chromatography and their structures were determined by linkage-specific sequential exoglycosidase digestion. These results demonstrated that recombinant APP has bi- and triantennary complex type N-glycans with fucosylated and nonfucosylated trimannosyl cores. In a similar fashion, the 2AB-labeled O-glycans derived from APP were determined to be mono- and disialylated core type 1 structures. Taken together, these results indicate that recombinant APP has sialylated bi- and triantennary N-glycans with fucosylated and nonfucosylated cores and sialylated O-glycans with core type 1 structures.

The role of insulin (INS) and insulin-like growth factor-I (IGF-I) in regulating human erythropoiesis. Studies in vitro under serum-free conditions--comparison to other cytokines and growth factors.

The role of insulin (INS), and insulin-like growth factor-I (IGF-I) in the regulation of human erythropoiesis is not completely understood. To address this issue we employed several complementary strategies including: serum free cloning of CD34+ cells, RT-PCR, FACS analysis, and mRNA perturbation with oligodeoxynucleotides (ODN). In a serum-free culture model, both INS and IGF-I enhanced survival of CD34+ cells, but neither of these growth factors stimulated their proliferation. The influence of INS and IGF-I on erythroid colony development was dependent on a combination of growth factors used for stimulating BFU-E growth. When BFU-E growth was optimally stimulated with erythropoietin (EpO) + kit ligand (KL) the large erythroid colonies developed normally even in the absence of INS or IGF-I. However, the addition of both of these growth factors slightly enhanced colony size. On the other hand, if erythroid colonies were stimulated suboptimally with EpO + IL-3 only, INS or IGF-I increased the number of small erythroid bursts by approximately 30%. Both INS and IGF-I activated signal transduction in maturing human erythropoietic cells as determined by phosphorylation of the insulin receptor substrate-2 (IRS-2) protein. We also found by RT-PCR that mRNA coding for INS-R is expressed in FACS sorted CD34+, c-kit-R+ marrow cells, and in cells isolated from BFU-E and CFU-GM colonies. Expression of INS-R protein on these cells was subsequently confirmed by cytofluorometry. In contrast, the receptor for insulin-like growth factor-I (IGF-IR) was not detected on CD34+ cells, and was first easily detectable on more differentiated cells derived from day 6 BFU-E and CFU-GM colonies. We conclude that INS and IGF-I may be survival factors for human CD34+ cells, but are not required during early erythropoiesis. In contrast, both growth factors may play some role at the final stages of erythroid maturation.

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein.

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N-glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.

Expression and analysis of presenilin 1 in a human neuronal system: localization in cell bodies and dendrites.

Mutations in the recently identified presenilin 1 gene on chromosome 14 cause early onset familial Alzheimer disease (FAD). Herein we describe the expression and analysis of the protein coded by presenilin 1 (PS1) in NT2N neurons, a human neuronal model system. PS1 was expressed using recombinant Semliki Forest virions and detected by introduced antigenic tags or antisera to PS1-derived peptides. Immunoprecipitation revealed two major PS1 bands of approximately 43 and 50 kDa, neither of which were N-glycosylated or O-glycosylated. Immunoreactive PS1 was detected in cell bodies and dendrites of NT2N neurons but not in axons or on the cell surface. PS1 was also detected in BHK cells, where it was also intracellular and colocalized with calnexin, a marker for the rough endoplasmic reticulum. A mutant form of PS1 linked to FAD did not differ from the wild-type protein at the light microscopic level. The model system described here will enable studies of the function of PS1 in human neurons and the role of mutant PS1 in FAD.

Purification of a secreted form of recombinant rabies virus glycoprotein: comparison of two affinity tags.

Expression of recombinant eukaryotic proteins in transfected mammalian cell lines has become an important approach for the characterization of the structure and function of these proteins. However, it is often difficult to recover and purify the recombinant proteins. Therefore, the use of fusion proteins incorporating epitope or affinity tags has become more widespread. In this paper, we directly compare two affinity tags, the hexahistidyl tag and the biotin peptide mimetic, Strep-tag, for use in purification of a recombinant soluble form of rabies virus glycoprotein secreted by transfected Chinese hamster ovary fibroblasts. The recombinant rabies virus glycoproteins are denoted RGP(WT)T441his and RGP(WT)T443s-tag, respectively. These affinity tags were chosen because the chromatographic matrices (Ni(II)-NTA-agarose and recombinant core streptavidin-agarose, respectively) were readily available and these methods offered the possibility of a one-step purification using mild elution conditions. However, in our hands, neither method allowed for a one-step purification protocol. Nonetheless, it was possible to purify RGP(WT)T441his to homogeneity from crude conditioned medium using a combination of metal-chelate affinity chromatography and immunoaffinity chromatography. In contrast, although the Strep-tag has been useful for purifying recombinant proteins expressed in bacteria, we were not able to effectively purify RGP(WT)T443s-tag from conditioned medium using chromatography on recombinant core streptavidin-agarose.

Purification of lysosomal arylsulfatase B from rat liver and its reactivity with lectins in affinity immunoelectrophoresis.

1. Arylsulfatase B (ASB) from lysosomal fraction of rat liver were isolated and purified 260-fold with a recovery of about 5%. 2. The enzyme in gradient PAGE 4-30% followed by immunoelectrophoresis migrated as a single peak of M(r) 84,000. The pI, measured by isoelectrofocusing in agarose followed by immunoelectrophoresis, was equal to 6.7. 3. ASB reacted with Con A, LCA, PSA, LTL, WGA, RCAI and did not react with PHA, SBA, HPA, CAA and PAL in crossed affino-immunoelectrophoresis or rocket immunoelectrophoresis. These results permit of preliminary elucidation of ASB glycan structure.