Immunoaffinity chromatography on antibodies immobilized on nitrocellulose powder.

A method is described for the use of nitrocellulose powder as a solid phase in a chromatographic procedure, for the immunoaffinity isolation of proteins. Two different immunoglobulins (Igs), anti-Datura innoxia lectin and anti-tyrosinase, were coupled to particulate nitrocellulose. A single step was then needed for purification to homogeneity of both D. innoxia lectin and mouse tyrosinase. Chaotropic and acidic agents proved to be effective in eluting antigens from Ig-nitrocellulose columns. The binding capacity of particulate nitrocellulose was around 3 mg Ig per milliliter of nitrocellulose, while the purification yields of the two proteins investigated under various eluting conditions were higher than 75%. The applicability of this method in the identification of metabolically labeled proteins in crude extracts is also demonstrated. Purification of proteins by affinity chromatography on their specific Igs linked to nitrocellulose matrices could be performed in both batch and column. The major advantages of this new method for purification of proteins are its rapidity, the reusability of the affinity matrices, and the high yields of purified protein obtained. The method could be seen as an alternative to the widely used immunoprecipitation technique.

Antiviral effect of N-butyldeoxynojirimycin against bovine viral diarrhea virus correlates with misfolding of E2 envelope proteins and impairment of their association into E1-E2 heterodimers.

The iminosugar N-butyldeoxynojirimycin (NB-DNJ), an endoplasmic reticulum alpha-glucosidase inhibitor, has an antiviral effect against bovine viral diarrhea virus (BVDV). In this report, we investigate the molecular mechanism of this inhibition by studying the folding pathway of BVDV envelope glycoproteins in the presence and absence of NB-DNJ. Our results show that, while the disulfide-dependent folding of E2 glycoprotein occurs rapidly (2.5 min), the folding of E1 occurs slowly (30 min). Both BVDV envelope glycoproteins associate rapidly with calnexin and dissociate with different kinetics. The release of E1 from the interaction with calnexin coincides with the beginning of E1 and E2 association into disulfide-linked heterodimers. In the presence of NB-DNJ, the interaction of E1 and E2 with calnexin is prevented, leading to misfolding of the envelope glycoproteins and inefficient formation of E1-E2 heterodimers. The degree of misfolding and the lack of association of E1 and E2 into disulfide-linked complexes in the presence of NB-DNJ correlate with the dose-dependent antiviral effect observed for this iminosugar.

Protein specific N-glycosylation of tyrosinase and tyrosinase-related protein-1 in B16 mouse melanoma cells.

Tyrosinase and tyrosinase-related protein-1 (TRP-1) are two melanogenic enzymes that regulate melanin biosynthesis. Both are glycoproteins and belong to the TRP-1 gene family. They share a significant level of sequence similarity in several regions, including the catalytic domain and the potential N-glycosylation sites. We have recently shown that inhibition of the early steps of N-glycan processing in B16F1 cells dramatically affects tyrosinase activity and melanin synthesis. We present here results on N-glycan processing of TRP-1 and tyrosinase and compare the maturation process and activity of both glycoproteins in the presence of inhibitors of the endoplasmic reticulum stages of N-glycosylation. N-glycan analysis reveals that each of these two glycoproteins contains a mixture of high-mannose and sialylated complex N-glycans. However, in contrast to TRP-1, tyrosinase presents a homogeneous high-mannose glycoform, also. In the presence of alpha-glucosidases inhibitors, the maturation of tyrosinase N-glycans is completely inhibited, whereas TRP-1 is still able to acquire some complex glycans, indicating that endomannosidase acts preferentially on the later glycoprotein. In addition, the dopa-oxidase activity of tyrosinase is totally abolished, whereas for TRP-1 it is only partially affected. The results suggest that despite their structural similarity, tyrosinase is more sensitive than TRP-1 to perturbations of early N-glycan processing, in terms of maturation and catalytical activity.

Study of the mechanism of antiviral action of iminosugar derivatives against bovine viral diarrhea virus.

The glucose-derived iminosugar derivatives N-butyl- and N-nonyl-deoxynojirimycin (DNJ) have an antiviral effect against a broad spectrum of viruses including Bovine viral diarrhea virus (BVDV). For BVDV, this effect has been attributed to the reduction of viral secretion due to an impairment of viral morphogenesis caused by the ability of DNJ-based iminosugar derivatives to inhibit ER alpha-glucosidases (N. Zitzmann, A. S. Mehta, S. Carrouée, T. D. Butters, F. M. Platt, J. McCauley, B. S. Blumberg, R. A. Dwek, and T. M. Block, Proc. Natl. Acad. Sci. USA 96:11878-11882, 1999). Here we present the antiviral features of newly designed DNJ derivatives and report for the first time the antiviral activity of long-alkyl-chain derivatives of deoxygalactonojirimycin (DGJ), a class of iminosugars derived from galactose which does not inhibit endoplasmic reticulum (ER) alpha-glucosidases. We demonstrate the lack of correlation between the ability of long-alkyl-chain DNJ derivatives to inhibit ER alpha-glucosidases and their antiviral effect, ruling out ER alpha-glucosidase inhibition as the sole mechanism responsible. Using short- and long-alkyl-chain DNJ and DGJ derivatives, we investigated the mechanisms of action of these drugs. First, we excluded their potential action at the level of the replication, protein synthesis, and protein processing. Second, we demonstrated that DNJ derivatives cause both a reduction in viral secretion and a reduction in the infectivity of newly released viral particles. Long-alkyl-chain DGJ derivatives exert their antiviral effect solely via the production of viral particles with reduced infectivity. We demonstrate that long-alkyl-chain DNJ and DGJ derivatives induce an increase in the quantity of E2-E2 dimers accumulated within the ER. The subsequent enrichment of these homodimers in secreted virus particles correlates with their reduced infectivity.

Tyrosinase folding and copper loading in vivo: a crucial role for calnexin and alpha-glucosidase II.

Tyrosinase is the key enzyme of melanin biosynthesis. It is a multiply glycosylated metalloenzyme, which has a long maturation time making it an ideal in vivo model system to probe protein folding and metal loading events. The use of NB-DNJ, an alpha-glucosidase I and II inhibitor has allowed us to dissect these processes. Here we show that tyrosinase folds through several inactive intermediates, at least two of which are recognised by the ER chaperone, calnexin. If the association with calnexin is prevented, more rapid folding occurs, the resulting protein fails to bind copper and is inactive. If dissociation from calnexin is inhibited, folding is prevented; the protein does not go through the normal secretory pathway and is targeted for degradation. Thus, tyrosinase folds off calnexin, giving alpha-glucosidase II a critical role, but the association with calnexin is essential to promote the correct folding which enables it to acquire copper.

Mutations at critical N-glycosylation sites reduce tyrosinase activity by altering folding and quality control.

Tyrosinase is a copper-containing enzyme that regulates melanin biosynthesis in mammals. Mutations at a single N-glycosylation sequon of tyrosinase have been reported to be responsible for oculocutaneous albinism type IA in humans, characterized by inactive tyrosinase and the total absence of pigmentation. To probe the role that each N-glycosylation site plays in the synthesis of biologically active tyrosinase, we analyzed the calnexin mediated folding of tyrosinase N-glycosylation mutants. We have determined that four of the six potential N-glycosylation sites, including that associated with albinism, are occupied. Analysis of the folding pathway and activity of 15 tyrosinase mutants lacking one or more of the occupied N-glycosylation sites shows that glycans at any two N-glycosylation sites are sufficient to interact with calnexin and give partial activity, but a specific pair of sites (Asn(86) and Asn(371)) is required for full activity. The mutants with less than two N-glycosylation sites do not interact with calnexin and show a complete absence of enzyme activity. Copper analysis of selected mutants suggests that the observed partial activity is due to two populations with differential copper content. By correlating the degree of folding with the activity of tyrosinase, we propose a local folding mechanism for tyrosinase that can explain the mechanism of inactivation of tyrosinase N-glycosylation mutants found in certain pigmentation disorders.

Investigation of the intracellular transport of tyrosinase and tyrosinase related protein (TRP)-1. The effect of endoplasmic reticulum (ER)-glucosidases inhibition.

Melanin biosynthesis is completely inhibited in the B16 melanoma cells following their incubation with inhibitors of the two ER glucosidases. This is primarily due to the inactivation of tyrosinase. Under the same conditions, the DOPA-oxidase activity of TRP-1 was only partially affected. In this report we investigate the effects of the perturbation of N-glycan processing in ER on the transport and activation of tyrosinase and TRP-1. We have localized the DOPA-oxidase activity in normal and inhibited cells and suggest that the first DOPA-reactive compartment of the secretory pathway (trans Golgi network) is also the site of tyrosinase activation. The inhibition of N-glycan processing does not affect the intracellular trafficking of the two melanogenic enzymes that are correctly transported to melanosomes. Immunoprecipitation experiments followed by analysis in SDS-PAGE under non-reducing conditions suggest that in inhibited cells, both tyrosinase and TRP-1 are synthesized in a modified conformation as compared to the normal proteins. These data suggest that the inhibition of melanin synthesis is not due to a defective transport but rather to conformational changes induced in the structure of tyrosinase and TRP-1 during their transit through the ER.