N-Linked Protein Glycosylation in the Endoplasmic Reticulum

The attachment of glycans to asparagine residues of proteins is an abundant and highly conserved essential modification in eukaryotes. The N-glycosylation process includes two principal phases: the assembly of a lipid-linked oligosaccharide (LLO) and the transfer of the oligosaccharide to selected asparagine residues of polypeptide chains. Biosynthesis of the LLO takes place at both sides of the endoplasmic reticulum (ER) membrane and it involves a series of specific glycosyltransferases that catalyze the assembly of the branched oligosaccharide in a highly defined way. Oligosaccharyltransferase (OST) selects the Asn-X-Ser/Thr consensus sequence on polypeptide chains and generates the N-glycosidic linkage between the side-chain amide of asparagine and the oligosaccharide. This ER-localized pathway results in a systemic modification of the proteome, the basis for the Golgi-catalyzed modification of the N-linked glycans, generating the large diversity of N-glycoproteome in eukaryotic cells. This article focuses on the processes in the ER. Based on the highly conserved nature of this pathway we concentrate on the mechanisms in the eukaryotic model organism Saccharomyces cerevisiae.The presence of glycans on proteins is known to influence their stability and solubility and the glycan core can contribute to folding processes (Shental-Bechor and Levy 2008; Hanson et al. 2009; Culyba et al. 2011). N-glycans also influence the function and activity of proteins (Skropeta 2009). The terminal residues of N-glycans play a key role in the quality control of protein folding in the ER. Ultimately the glycan signals whether a protein is correctly folded and can leave the ER to continue its maturation in the Golgi or whether the protein is not correctly folded and is degraded (Helenius and Aebi 2004; Aebi et al. 2010). It is therefore of great importance that the oligosaccharide to be transferred to proteins is complete. This “quality control” of the oligosaccharide is mediated by the substrate specificity of oligosaccharyltransferase.     

N-Linked Glycosylation in Archaea: a Structural,Functional, and Genetic Analysis

SUMMARY

N-glycosylation of proteins is one of the most prevalent posttranslational modifications in nature. Accordingly, a pathway with shared commonalities is found in all three domains of life. While excellent model systems have been developed for studying N-glycosylation in both Eukarya and Bacteria, an understanding of this process in Archaea was hampered until recently by a lack of effective molecular tools. However, within the last decade, impressive advances in the study of the archaeal version of this important pathway have been made for halophiles, methanogens, and thermoacidophiles, combining glycan structural information obtained by mass spectrometry with bioinformatic, genetic, biochemical, and enzymatic data. These studies reveal both features shared with the eukaryal and bacterial domains and novel archaeon-specific aspects. Unique features of N-glycosylation in Archaea include the presence of unusual dolichol lipid carriers, the use of a variety of linking sugars that connect the glycan to proteins, the presence of novel sugars as glycan constituents, the presence of two very different N-linked glycans attached to the same protein, and the ability to vary the N-glycan composition under different growth conditions. These advances are the focus of this review, with an emphasis on N-glycosylation pathways in Haloferax, Methanococcus, and Sulfolobus.     

Role of N-Linked Glycosylation of the 5-HT2A Receptor in JC Virus Infection

JC virus (JCV) is a human polyomavirus and the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection of host cells is dependent on interactions with cell surface asparagine (N)-linked sialic acids and the serotonin 5-hydroxytryptamine_2A receptor (5-HT_2AR). The 5-HT_2AR contains five potential N-linked glycosylation sites on the extracellular N terminus. Glycosylation of other serotonin receptors is essential for expression, ligand binding, and receptor function. Also, glycosylation of cellular receptors has been reported to be important for JCV infection. Therefore, we hypothesized that the 5-HT_2AR N-linked glycosylation sites are required for JCV infection. Treatment of 5-HT_2AR-expressing cells with tunicamycin, an inhibitor of N-linked glycosylation, reduced JCV infection. Individual mutation of each of the five N-linked glycosylation sites did not affect the capacity of 5-HT_2AR to support JCV infection and did not alter the cell surface expression of the receptor. However, mutation of all five N-linked glycosylation sites simultaneously reduced the capacity of 5-HT_2AR to support infection and altered the cell surface expression. Similarly, tunicamycin treatment reduced the cell surface expression of 5-HT_2AR. Mutation of all five N-linked glycosylation sites or tunicamycin treatment of cells expressing wild-type 5-HT_2AR resulted in an altered electrophoretic mobility profile of the receptor. Treatment of cells with PNGase F, to remove N-linked oligosaccharides from the cell surface, did not affect JCV infection in 5-HT_2AR-expressing cells. These data affirm the importance of 5-HT_2AR as a JCV receptor and demonstrate that the sialic acid component of the receptor is not directly linked to 5-HT_2AR.The initial interaction between virus and host occurs via molecular interactions of viral attachment proteins and receptors on host cells. Therefore, receptor recognition is a critical host cell determinant and may play a key regulatory role in viral pathogenesis. The polyomavirus JC virus (JCV) is a ubiquitous human pathogen (21, 25, 32) that is initially subclinical yet establishes a persistent infection in the kidney (11). In immunosuppressed individuals JCV can become reactivated, leading to infection in the central nervous system (CNS) (13-15, 20), where the virus specifically targets glial cells, including astrocytes and the myelin-producing cells, oligodendrocytes (40, 48). JCV infection and cytolytic destruction of oligodendroglia cause the fatal disease progressive multifocal leukoencephalopathy (PML) (1, 22). The most common cause of PML is associated with human immunodeficiency virus (HIV) and AIDS (10, 23). However, in recent years PML has been reported in patients receiving immunosuppressive therapies for autoimmune diseases such as Crohn''s disease (44), multiple sclerosis (MS) (24, 26, 28, 47), systemic lupus erythematosus (5, 33), and rheumatoid arthritis (5, 19, 37). The prognosis of PML is bleak, as the disease progresses rapidly and usually proves fatal within 1 year of the onset of symptoms. While current treatment options for PML are limited (23), recent studies suggest that mirtazapine, a serotonin receptor antagonist, may be capable of slowing the progression of PML (6, 27, 45, 46).JCV has a nonenveloped, icosahedral capsid that encapsidates a circular double-stranded DNA (dsDNA) genome (39). JCV attachment to cells is mediated by an N-linked glycoprotein with either α(2,3)- or α(2,6)-linked sialic acid (16, 31), suggesting that N-linked glycosylation of cellular receptors is important for JCV infection. N-linked glycosylation is a posttranslational process by which oligosaccharides are added to asparagine residues, and this modification is important for protein processing, folding, expression, and function (43). Previous studies from our laboratory revealed that the JCV also requires the serotonin 5-hydroxytryptamine_2A receptor (5-HT_2AR) to mediate JCV infection (18, 35, 38), while others report that JCV infection can occur in the absence of 5-HT_2AR (7, 8). 5-HT_2AR is a seven-transmembrane-spanning G-protein-coupled receptor that belongs to a large family of 5-HT serotonin receptors. 5-HT_2AR is abundantly expressed on cells in the brain (4), including glial cells (3), and in the kidney (4), which parallels the sites of JCV infection. N-linked glycosylation plays a key regulatory role in the function of serotonin receptors. Mutation of N-linked glycosylation sites in human 5-HT_3AR and 5-HT_5AR results in decreased expression at the plasma membrane, which is critical for receptor function (17, 34). N-linked glycosylation of murine 5-HT_3AR regulates plasma membrane targeting, ligand binding, Ca²⁺ flux, and receptor trafficking (36), suggesting that glycosylation is essential for expression and function of serotonin receptors.While previous studies have concluded that JCV utilizes an N-linked glycoprotein with α(2,3)-linked sialic acid (31) or α(2,6)-linked sialic acid (16) and 5-HT_2AR (18) to initiate infection in host cells, the mechanism(s) by which JCV engages its cellular receptors and the importance of receptor glycosylation remain unclear. 5-HT_2AR contains potential asparagine (N)-linked glycosylation sites, five of which are predicted to be expressed in the extracellular amino-terminal region, where they could be accessible to the virus (2). The goal of this study was to determine whether potential N-linked glycosylation sites expressed in 5-HT_2AR are required for JCV infection. We found that N-linked glycosylation of 5-HT_2AR is important for receptor expression but not necessary for JCV infection.     

Characterization of N-Linked Protein Glycosylation in Helicobacter pullorum

The first bacterial N-linked glycosylation system was discovered in Campylobacter jejuni, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologues are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria, including three Helicobacter species: H. pullorum, H. canadensis, and H. winghamensis. In contrast to C. jejuni, in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli. Analysis of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum, PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the −2 position of the N-glycosylation sequon, as for C. jejuni. Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.Glycosylation is one of the most common protein modifications, and eukaryotes glycosylate many of their secreted proteins with asparagine or N-linked glycans. This process is thought to have diverse roles in protein folding, quality control, protein secretion, and sorting (13). Eukaryotic glycosylation takes place at the luminal side of the endoplasmic reticulum (ER) membrane, where a preassembled oligosaccharide is transferred from a lipid carrier to asparagine residues within an N-X-S/T consensus sequence, where X can be any amino acid except proline (19). The coupling of glycan to the protein takes place cotranslationally as nascent polypeptide chains cross the ER membrane via a translocon apparatus (5). This reaction involves a protein complex of at least eight subunits (49), with the STT3 protein (50, 52) apparently acting as the central enzyme in the process of N-linked protein glycosylation (29, 48). The STT3 protein consists of an amino terminus with multiple membrane-spanning domains and a carboxy-terminal region containing the highly conserved WWDYG amino acid sequence motif (15).The first prokaryotic glycoproteins were described for archaeal species over 30 years ago (26), and for some time it was thought that protein glycosylation was a eukaryotic and archaeal, but not a bacterial, trait. However, there are now many examples of protein glycosylation in species from the domain Bacteria. For example, general O-linked protein glycosylation systems in which functionally diverse sets of proteins are glycosylated via a single pathway have recently been identified in Neisseria and Bacteroides spp. (8, 21, 44). The most-well-characterized bacterial species with respect to protein glycosylation is the enteropathogen Campylobacter jejuni, which encodes an O-linked system that glycosylates the flagellin protein of the flagellar filament along with the first described bacterial N-linked glycosylation system (39).The C. jejuni N-linked glycosylation pathway is encoded by genes from a single protein glycosylation, or pgl, locus (38). The glycosylation reaction is thought to occur at the periplasmic face of the bacterial inner membrane mediated by the product of the STT3 orthologue pglB (46). The C. jejuni heptasaccharide glycan is assembled on a lipid carrier in the cytoplasm through the action of glycosyltransferases encoded by the pglA, pglC, pglH, pglJ, and pglI genes (11, 12, 24, 31). This lipid-linked oligosaccharide (LLO) is then “flipped” into the periplasm by the pglK gene product, or “flippase” (1), and transferred by PglB onto an asparagine residue within an extended D/E-X-N-X-S/T sequon (19). Many C. jejuni periplasmic and surface proteins of diverse function are N glycosylated (51), yet the function of glycosylation remains elusive. Unlike in eukaryotes, this process occurs posttranslationally, and the surface location of the sequon in folded proteins appears to be required for glycosylation (20).The C. jejuni pgl gene locus can be transferred into Escherichia coli, and the corresponding gene products will function to transfer the heptasaccharide onto asparagine residues of coexpressed C. jejuni glycoproteins as well as non-C. jejuni proteins containing the appropriately located acceptor sequon (19, 46). When alternative lipid-linked glycans are present, such as those involved in lipopolysaccharide biosynthesis, glycans with diverse structure can also be transferred onto proteins (7). Although there are limitations, particularly with regard to the apparent structural requirement for an acetamido group on the C-2 carbon of the reducing end sugar (7, 47), this is still a significant advance toward tractable in vivo systems for glycoconjugate synthesis. The identification and characterization of further bacterial PglB proteins with potentially diverse properties would considerably expand the utility of such systems. Data from genome sequencing indicate that pglB orthologues are found in species closely related to C. jejuni, such as Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis (40), as well as in the more distantly related species Wolinella succinogenes (2). These species are members of the phylogenetic grouping known as the epsilon subdivision of the Proteobacteria, or Epsilonproteobacteria, consisting of the well-established genera Campylobacter, Helicobacter, Arcobacter, and Wolinella, which are often associated with human and animal hosts, as well as a number of newly recognized groupings of environmental bacteria often found in sulfidic environments (3). However, not all species of Epsilonproteobacteria contain pglB orthologues, and until recently, all characterized Helicobacter species lacked pglB genes.Given the considerable interest in exploiting bacterial protein glycosylation, especially the C. jejuni N-linked glycosylation system, for generating glycoconjugates of biotechnological and therapeutic potential, the functional characterization of newly discovered pglB orthologues is a priority. In this report we describe the application of an in vitro oligosaccharyltransferase assay to investigate N-linked glycosylation initially in C. jejuni, where the utility of this approach was demonstrated, and then in Helicobacter pullorum, demonstrating that one of the two H. pullorum PglB enzymes is responsible for N-linked protein glycosylation with a pentasaccharide glycan.     

Contributions of N-Linked Glycosylation to the Expression of a Functional α7-Nicotinic Receptor in Xenopus Oocytes

Abstract: The α7 subunit of the neuronal nicotinic acetylcholine receptor, when expressed in Xenopus oocytes, forms homooligomeric ligand-gated ion channels that are blocked by a snake toxin, α-bungarotoxin. The amino-terminal extracellular domain of the α7 sequence has three consensus sites for asparagine-linked glycosylation (N46DS, N90MS, and N133AS). In this study, we show that α7 expressed either in vivo or in vitro is a glycoprotein of 57 kDa. In addition, we demonstrate by site-directed mutagenesis that all three consensus sites are used for glycosylation. To elucidate the role(s) of asparagine-linked glycosylation in the formation and function of the α7 receptor, wild-type and glycosylation-deficient α7 subunits were expressed in COS cells and oocytes. We examined biochemical and physiological properties of expressed receptors and found that α7 glycosylation mutations do not affect homooligomerization and surface protein expression of the α7 receptor but do affect surface expression of α-bungarotoxin binding sites and the function of the receptor. Our data indicate that asparagine-linked glycosylation is required for the expression of a functional α7 receptor in oocytes.     

Role for Calnexin and N-Linked Glycosylation in the Assembly and Secretion of Hepatitis B Virus Middle Envelope Protein Particles

Unlike those of the S and the L envelope proteins, the functional role of the related M protein in the life cycle of the hepatitis B virus (HBV) is less understood. We now demonstrate that a single N glycan, specific for M, is required for efficient secretion of M empty envelope particles. Moreover, this glycan mediates specific association of M with the chaperone calnexin. Conversely, the N glycan, common to all three envelope proteins, is involved neither in calnexin binding nor in subviral particle release. As proper folding and trafficking of M need the assistance of the chaperone, the glycan-dependent association of M with calnexin may thus play a crucial role in the assembly of HBV. Beyond being modified by N glycosylation, M is modified by O glycosylation occurring within its amino acid sequence at positions 27 to 47. The O glycans, however, were found to be dispensable for secretion of M but may rather support viral infectivity. Surprisingly, nonglycosylated M localizes exclusively to the cytosol, either for degradation or for a yet-unknown function.     

A Functional and Putative Physiological Role of Calcitriol in Patched1/Smoothened Interaction

The Patched1 (Ptch)-mediated inhibition of Smoothened (Smo) is still an open question. However, a direct Ptch/Smo interaction has been excluded, Smo modulators were identified, but the endogenous signal transmitting molecule remains undiscovered. Here, we demonstrate that calcitriol, the hormonally active form of vitamin D₃, is an excellent candidate for transmission of Ptch/Smo interaction. Our study reveals that Ptch expression is sufficient to release calcitriol from the cell and that calcitriol inhibits Smo action and ciliary translocation by acting on a site distinct from the 7-transmembrane domain or the cysteine-rich domain. Moreover calcitriol strongly synergizes with itraconazole (ITZ) in Smo inhibition, which did not result from elevated calcitriol bioavailability due to ITZ-mediated 24-hydroxylase inhibition but rather from a direct interaction of the compounds at the level of Smo. Together, we suggest that calcitriol represents a possible endogenous transmitter of Ptch/Smo interaction. Moreover calcitriol or calcitriol derivatives combined with ITZ might be a treatment option of Hedgehog-associated cancers.     

Functional Role of BSL1 Subcellular Localization in Brassinosteroid Signaling

The bri1 Suppressor 1 (BSU1) family mediates the brassinosteroid (BR) signal transduction pathway that orchestrates a wide range of developmental and physiological responses in plants. In Arabidopsis, BSU1 family members (BSU1, BSL1, BSL2, and BSL3) enhance BR signaling through hetero- and homo-oligomerization. Interestingly, BSL1 localizes in the cytoplasm whereas the other three homologs occur in both the nucleus and the cytoplasm. However, little is known about whether differential subcellular localization of BSL1 affects oligomerization of BSU1 family members or modulates BR signaling. Here we show that homooligomeric BSL1 forms cytoplasmic puncta and oligomeric combinations between BSU1 family members determine their subcellular localization. We demonstrate that BSL1 has a distinct role in regulating BSL2 and BSL3 through cytoplasmic oligomerization. Overexpression of BSL1 reduced nuclear accumulation of BSL2 and BSL3. Furthermore, mutagenic analysis indicates that nuclear localization of BSL1 promotes BR signaling, suggesting that BSL1 plays a functional role modulating BR signaling through cytoplasmic retention of BSL2 and BSL3.     

Functional Adaptation in Female Rats: The Role of Estrogen Signaling

Background

Sex steroids have direct effects on the skeleton. Estrogen acts on the skeleton via the classical genomic estrogen receptors alpha and beta (ERα and ERβ), a membrane ER, and the non-genomic G-protein coupled estrogen receptor (GPER). GPER is distributed throughout the nervous system, but little is known about its effects on bone. In male rats, adaptation to loading is neuronally regulated, but this has not been studied in females.

Methodology/Principal Findings

We used the rat ulna end-loading model to induce an adaptive modeling response in ovariectomized (OVX) female Sprague-Dawley rats. Rats were treated with a placebo, estrogen (17β-estradiol), or G-1, a GPER-specific agonist. Fourteen days after OVX, rats underwent unilateral cyclic loading of the right ulna; half of the rats in each group had brachial plexus anesthesia (BPA) of the loaded limb before loading. Ten days after loading, serum estrogen concentrations, dorsal root ganglion (DRG) gene expression of ERα, ERβ, GPER, CGRPα, TRPV1, TRPV4 and TRPA1, and load-induced skeletal responses were quantified. We hypothesized that estrogen and G-1 treatment would influence skeletal responses to cyclic loading through a neuronal mechanism. We found that estrogen suppresses periosteal bone formation in female rats. This physiological effect is not GPER-mediated. We also found that absolute mechanosensitivity in female rats was decreased, when compared with male rats. Blocking of adaptive bone formation by BPA in Placebo OVX females was reduced.

Conclusions

Estrogen acts to decrease periosteal bone formation in female rats in vivo. This effect is not GPER-mediated. Gender differences in absolute bone mechanosensitivity exist in young Sprague-Dawley rats with reduced mechanosensitivity in females, although underlying bone formation rate associated with growth likely influences this observation. In contrast to female and male rats, central neuronal signals had a diminished effect on adaptive bone formation in estrogen-deficient female rats.     

Hrs Promotes Ubiquitination and Mediates Endosomal Trafficking of Smoothened in Drosophila Hedgehog Signaling

In Hedgehog (Hh) signaling, the seven-transmembrane protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation, ubiquitination, and cell surface accumulation. However, it is not clear how Smo cell surface accumulation and intracellular trafficking are regulated. Here, we demonstrate that inactivation of Hrs by deletion or RNAi accumulates Smo in the late endosome that is marked by late endosome markers. Inactivation of Hrs enhances the wing defects caused by dominant-negative Smo. We show that Hrs promotes Smo ubiquitination, deleting the ubiquitin-interacting-motif (UIM) in Hrs abolishes the ability of Hrs to regulate Smo ubiquitination. However, the UIM domain neither recognizes the ubiquitinated Smo nor directly interacts with Smo. Hrs lacking UIM domain still downregulates Smo activity even though to a less extent. We have characterized that the N-terminus of Hrs directly interacts with the PKA/CK1 phosphorylation clusters to prevent Smo phosphorylation and activation, indicating an ubiquitin-independent regulation of Smo by Hrs. Finally, we found that knockdown of Tsg101 accumulates Smo that is co-localized with Hrs and other late endosome markers. Taken together, our data indicate that Hrs mediates Smo trafficking in the late endosome by not only promoting Smo ubiquitination but also blocking Smo phosphorylation.     

表皮生长因子样重复序列糖基化对Notch信号通路的作用

Notch受体是一类存在于多细胞生物中进化上高度保守的跨膜蛋白受体,其介导的信号通路在组织器官的生长发育中起重要作用。糖基化修饰是一类重要的影响多信号通路的蛋白翻译后修饰。Notch受体胞外域的表皮生长因子样重复序列（EGF-R）存在多种糖基化修饰如O-葡聚糖、O-岩藻糖聚糖、O- N-乙酰氨基葡萄糖,这些糖基化修饰可以促进或抑制Notch信号通路。本文主要阐述表皮生长因子样重复序列（EGF-R）的主要糖基化以及对Notch信号通路的影响和其异常导致相关的疾病。     

Action Mechanisms of the Secondary Metabolite Euplotin C: Signaling and Functional Role in Euplotes

ABSTRACT. Among secondary metabolites, the acetylated hemiacetal sesquiterpene euplotin C has been isolated from the marine, ciliated protist Euplotes crassus, and provides an effective mechanism for reducing populations of potential competitors through its cytotoxic properties. However, intracellular signaling mechanisms and their functional correlates mediating the ecological role of euplotin C are largely unknown. We report here that, in E. vannus (an Euplotes morphospecies that does not produce euplotin C and shares with E. crasssus the same interstitial habitat), euplotin C rapidly increases the intracellular concentration of both Ca²⁺ and Na⁺, suggesting a generalized effect of this metabolite on cation transport systems. In addition, euplotin C does not induce oxidative stress, but modulates the electrical properties of E. vannus through an increase of the amplitude of graded action potentials. These events parallel the disassembling of the ciliary structures, the inhibition of cell motility, the occurrence of aberrant cytoplasmic vacuoles, and the rapid inhibition of phagocytic activity. Euplotin C also increases lysosomal pH and decreases lysosomal membrane stability of E. vannus. These results suggest that euplotin C exerts a marked disruption of those homeostatic mechanisms whose efficiency represents the essential prerequisite to face the challenges of the interstitial environment.     

Effect of N-Linked Glycosylation on Secretion, Activity, and Stability of α-Amylase from Aspergillus oryzae

The effect of N-linked glycosylation on secretion, activity, and stability of α-amylase from Aspergillus oryzae grown as dispersed filaments was studied. In the presence of tunicamycin the fungus grew either as dispersed filaments or as one large pellet, whereas growth was as dispersed filaments in all control cultures. The presence of tunicamycin affected neither biomass, level of secreted α-amylase, nor total amount of secreted protein in cultures growing as dispersed filaments. In these cultures both glycosylated and nonglycosylated α-amylase appeared in the culture medium as well as in the cells, whereas in control cultures only the glycosylated form of α-amylase was found in the medium and in the cells. The presence of nonglycosylated α-amylase in the medium seemed to result from active secretion rather than from autolysis of the mycelium or extracellular deglycosylation. Deglycosylation with Endo H of crude α-amylase in culture filtrate did not affect its stability towards heat, acid pH, or proteolytic degradation. Received: 22 December 1997 / Accepted: 24 February 1998