Inhibitory Effect of Resveratrol on Candida albicans Biofilm Formation.

Candida albicans is the primary candidiasis-causing fungal pathogen in humans, and one of its most important virulence factors is the ability to form biofilms. Moreover, these biofilms are often resistant to antifungal agents, so there is a need to develop alternative elimination strategies and therapeutic agents for such infections. The antifungal activity of resveratrol, a phytoalexin polyphenolic compound, impairs the morphological transition of C. albicans under various hypha-inducing conditions and inhibits growth of the yeast-form and mycelia. The purpose of this study was to investigate the effect of resveratrol against C. albicans biofilm formation. The developmental, sustained, and mature stages of biofilm formation were affected or inhibited by resveratrol. Exposure to resveratrol at the developmental stage inhibited growth of C. albicans in a dose-dependent manner. A >30% reduction was observed in sustained biofilm growth in the presence of 200 µg/ml resveratrol in comparison with in its absence. In terms of disruption of matured biofilm, 6.25-100 µg/ml resveratrol significantly reduced cell viability of C. albicans compared with in a control sample (p<0.05). The present results indicate that resveratrol has the potential to serve as an anti-Candida treatment and preventive tool which functions by inhibiting existing or under-forming C. albicans biofilms.

Biochemical Characterizations of the Putative Endolysin Ecd09610 Catalytic Domain from Clostridioides difficile.

Clostridioides difficile is the major pathogen of pseudomembranous colitis, and novel antimicrobial agents are sought after for its treatment. Phage-derived endolysins with species-specific lytic activity have potential as novel antimicrobial agents. We surveyed the genome of C. difficile strain 630 and identified an endolysin gene, Ecd09610, which has an uncharacterized domain at the N-terminus and two catalytic domains that are homologous to glucosaminidase and endopeptidase at the C-terminus. Genes containing the two catalytic domains, the glucosaminidase domain and the endopeptidase domain, were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. The purified domain variants showed lytic activity almost specifically for C. difficile, which has a unique peptide bridge in its peptidoglycan. This species specificity is thought to depend on substrate cleavage activity rather than binding. The domain variants were thermostable, and, notably, the glucosaminidase domain remained active up to 100 °C. In addition, we determined the optimal pH and salt concentrations of these domain variants. Their properties are suitable for formulating a bacteriolytic enzyme as an antimicrobial agent. This lytic enzyme can serve as a scaffold for the construction of high lytic activity mutants with enhanced properties.

Transesterification of phosphatidylcholine in sn-1 position through direct use of lipase-producing Rhizopus oryzae cells as whole-cell biocatalyst.

The enzymatic process presents an advantage of producing specified phospholipids that rarely exist in nature. In this study, we investigated the regiospecific modification of phosphatidylcholine (PC) in the sn-1 position using immobilized Rhizopus oryzae. In a reaction mixture containing egg yolk PC and exogenous lauric acid (LA) in n-hexane, lipase-producing R. oryzae cells immobilized within biomass support particles (BSPs) showed a much higher transesterification activity than lipase powders. To improve the product yield, several parameters including substrate ratio and reaction time were investigated, resulting in the incorporation of 44.2% LA into the product PC after a 48-h reaction. The analysis of the molecular structure showed that a large proportion of exogenous LA (>90%) was incorporated in the sn-1 position of the enzymatically modified PC. Moreover, the BSP-immobilized R. oryzae maintained its activity for more than 12 batch cycles. The presented results, therefore, suggest the applicability of BSP-immobilized R. oryzae as a whole-cell biocatalyst for the regiospecific modification of phospholipids.

Characterization of a novel murine preadipocyte line, AP-18, isolated from subcutaneous tissue: analysis of adipocyte-related gene expressions.

Adipocyte lines are a useful tool for adipocyte research. Recently, a new preadipocyte line designated AP-18 was established from subcutaneous tissue of the C3H/He mouse. In this study, we further characterized AP-18 cells. Adipocyte differentiation was assessed by accumulation of fat droplets stained by Oil Red O. The expression of the preadipocyte- or adipocyte-specific genes and adipocytokine genes was analysed qualitatively by RT-PCR and quantitatively by real-time PCR in comparison with the LM cell, a murine fibroblast line, and the 3T3-L1 cell, respectively. AP-18 cells were fibroblastoid in maintenance culture. After the confluence, fat droplets were accumulated in 50-60% of the cells cultured in the medium alone and in 70-90% of the cells cultured with insulin within 2 to 3 weeks. The fat accumulation was not promoted by the addition of dexamethazone, IBMX (3-isobutyl-1-methylxanthine) or troglitazone in combination with insulin, which were obligatory for differentiation of the 3T3-L1 cell, a murine preadipocyte line. Throughout the differentiation, AP-18 cells expressed Pref-1, LPL, C/EBP beta, C/EBP delta, RXR alpha, C/EBP alpha, PPAR gamma, RXR gamma, aP2, GLUT4, SCD1, UCP2, UCP3, TNFalpha, resistin, leptin, adiponectin and PAI-1 genes, but not the UCP1 gene, indicating that the cell is derived from WAT (white adipose tissue). The time course of these gene expressions was similar to that of 3T3-L1 cells, although the expressions were slower and lower in AP-18 cells. These data indicate that AP-18 cells are preadipocytes originated from WAT and differentiate into adipocytes under more physiological conditions than 3T3-L1 cells. AP-18 may be useful in adipocyte research.

Surfactant-modified yeast whole-cell biocatalyst displaying lipase on cell surface for enzymatic production of structured lipids in organic media.

The cell surface engineering system, in which functional proteins are genetically displayed on microbial cell surfaces, has recently become a powerful tool for applied biotechnology. Here, we report on the surfactant modification of surface-displayed lipase to improve its performance for enzymatic synthesis reactions. The lipase activities of the surfactant-modified yeast displaying Rhizopus oryzae lipase (ROL) were evaluated in both aqueous and nonaqueous systems. Despite the similar lipase activities of control and surfactant-modified cells in aqueous media, the treatment with nonionic surfactants increased the specific lipase activity of the ROL-displaying yeast in n-hexane. In particular, the Tween 20-modified cells increased the cell surface hydrophobicity significantly among a series of Tween surfactants tested, resulting in 8-30 times higher specific activity in organic solvents with relatively high log P values. The developed cells were successfully used for the enzymatic synthesis of phospholipids and fatty acid methyl esters in n-hexane, whereas the nontreated cells produced a significantly low yield. Our results thus indicate that surfactant modification of the cell surface can enhance the potential of the surface-displayed lipase for bioconversion.

Preparation and comparative characterization of immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase for enzymatic biodiesel production.

In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn-2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn-2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn-2 to the sn-1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.

Simultaneous conversion of free fatty acids and triglycerides to biodiesel by immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase.

The presence of high levels of free fatty acids (FFA) in oil is a barrier to one-step biodiesel production. Undesirable soaps are formed during conventional chemical methods, and enzyme deactivation occurs when enzymatic methods are used. This work investigates an efficient technique to simultaneously convert a mixture of free fatty acids and triglycerides (TAG). A partial soybean hydrolysate containing 73.04% free fatty acids and 24.81% triglycerides was used as a substrate for the enzymatic production of fatty acid methyl ester (FAME). Whole-cell Candida antarctica lipase B-expressing Aspergillus oryzae, and Novozym 435 produced only 75.2 and 73.5% FAME, respectively. Fusarium heterosporum lipase-expressing A. oryzae produced more than 93% FAME in 72 h using three molar equivalents of methanol. FFA and TAG were converted simultaneously in the presence of increasing water content that resulted from esterification. Therefore, F. heterosporum lipase with a noted high level of tolerance of water could be useful in the industrial production of biodiesel from feedstock that has high proportion of free fatty acids.

Lipase cocktail for efficient conversion of oils containing phospholipids to biodiesel.

The presence of phospholipid has been a challenge in liquid enzymatic biodiesel production. Among six lipases that were screened, lipase AY had the highest hydrolysis activity and a competitive transesterification activity. However, it yielded only 21.1% FAME from oil containing phospholipids. By replacing portions of these lipases with a more robust bioFAME lipase, CalT, the combination of lipase AY-CalT gave the highest FAME yield with the least amounts of free fatty acids and partial glycerides. A higher methanol addition rate reduced FAME yields for lipase DF-CalT and A10D-CalT combinations while that of lipase AY-CalT combination improved. Optimizing the methanol addition rate for lipase AY-CalT resulted in a FAME yield of 88.1% at 2h and more than 95% at 6h. This effective use of lipases could be applied for the rapid and economic conversion of unrefined oils to biodiesel.

VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells.

Neuropilin-1 (NRP1) has been identified as a VEGF-A receptor. DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1. In the present study, the RNA interference of VEGF-A or NRP1 suppressed DJM-1 cell proliferation. Furthermore, the overexpression of the NRP1 wild type restored shNRP1-treated DJM-1 cell proliferation, whereas NRP1 cytoplasmic deletion mutants did not. A co-immunoprecipitation analysis revealed that VEGF-A induced interactions between NRP1 and GIPC1, a scaffold protein, and complex formation between GIPC1 and Syx, a RhoGEF. The knockdown of GIPC1 or Syx reduced active RhoA and DJM-1 cell proliferation without affecting the MAPK or Akt pathway. C3 exoenzyme or Y27632 inhibited the VEGF-A-induced proliferation of DJM-1 cells. Conversely, the overexpression of the constitutively active form of RhoA restored the proliferation of siVEGF-A-treated DJM-1 cells. Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor. A cell-penetrating oligopeptide that targeted GIPC1/Syx complex formation inhibited the VEGF-A-induced activation of RhoA and suppressed DJM-1 cell proliferation. In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

Distribution of the MCS4 RNA genes in mycoplasmas belonging to the Mycoplasma mycoides cluster.

MCS4 RNA is one of the small stable RNAs found in Mycoplasma capricolum subsp. capricolum type strain California kid. This RNA has a sequence similarity to that of eukaryotic U6 snRNA. There are two genes encoding MCS4 RNA, designated mcs4a and mcs4b, in the genome. Homologous sequences of these genes were not found in databases of other bacterial sequences. We searched for MCS4 RNA and its genes in other bacteria by PCR and hybridization techniques. The results strongly suggested that this RNA exists only in a limited species of mycoplasmas belonging to the Mycoplasma mycoides cluster.

Stable expression of gastric proton pump activity at the cell surface.

Stable cell lines expressing the gastric proton pump alpha- and/or beta-subunits were constructed. The cell line co-expressing the alpha- and beta-subunits showed inward Rb(+) transport, which was activated by Rb(+) in a concentration-dependent manner. In the alpha+beta-expressing cell line, rapid recovery of intracellular pH was also observed after acid load, indicating that this cell line transported protons outward. These ion transport activities were inhibited by a proton pump inhibitor, 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080). In a membrane fraction of the alpha+beta-expressing cell line, K(+)-stimulated ATPase (K(+)-ATPase) activity and the acylphosphorylation of the alpha-subunit were observed, both of which were also inhibited by SCH 28080. The specific activity and properties of the K(+)-ATPase were comparable to those found in the native gastric proton pump. In the stable cell lines, the alpha-subunit was retained in the intracellular compartment and was unstable in the absence of the beta-subunit, but it was stabilized and reached the cell surface in the presence of the beta-subunit. On the other hand, the beta-subunit was stable and able to travel to the cell surface in the absence of the alpha-subunit. These cell lines are ideal for the structure-function study of ion transport by the gastric proton pump as well as for characterization of the cellular regulation of surface expression of the functional proton pump.

Expression of copper-transporting P-type adenosine triphosphatase in human esophageal carcinoma.

A major obstacle in the treatment of esophageal carcinoma is the intrinsic/acquired resistance to cisplatin-based chemotherapy. Copper-transporting P-type adenosine triphosphatase (ATP7B) has been reported to be associated with cisplatin resistance in vitro. However, the clinical significance of this transporter has not previously been addressed. Our goal was to investigate if ATP7B is expressed in esophageal carcinoma and whether its expression correlates with reduced responsiveness to cisplatin treatment. We retrospectively examined the expression of ATP7B in primary esophageal carcinoma and its association with chemotherapeutic effect. Tissues were surgically removed from 17 esophageal carcinoma patients. Twelve of them received cisplatin-based chemotherapy before surgery. We performed immunohistochemical analysis of ATP7B using a monoclonal antibody against ATP7B in 17 esophageal carcinomas. A variable degree of cytoplasmic staining of tumor cells was observed in 76.5% (13/17 cases) of the analyzed carcinomas. ATP7B expression was not observed in adjacent non-neoplastic tissues. ATP7B positivity was not significant in gender, age, histopathological grading or TNM categories. Patients with ATP7B-positive tumors tended to have an inferior response to chemotherapy compared with the patients with ATP7B-negative tumors. These findings suggest that overexpression of ATP7B in esophageal carcinoma could be associated with unfavorable clinical outcome in patients treated with cisplatin-based chemotherapy. Therefore, ATP7B gene expression might be considered as a chemoresistance marker for cisplatin in the patients of esophageal carcinoma and provider of important information on the strategy against esophageal carcinoma.

Enzymatic production of biodiesel from waste cooking oil in a packed-bed reactor: an engineering approach to separation of hydrophilic impurities.

An engineering approach was applied to an efficient biodiesel production from waste cooking oil. In this work, an enzymatic packed-bed reactor (PBR) was integrated with a glycerol-separating system and used successfully for methanolysis, yielding a methyl ester content of 94.3% and glycerol removal of 99.7%. In the glycerol-separating system with enhanced retention time, the effluent contained lesser amounts of glycerol and methanol than those in the unmodified system, suggesting its promising ability to remove hydrophilic impurities from the oil layer. The PBR system was also applied to oils with high acid values, in which fatty acids could be esterified and the large amount of water was extracted using the glycerol-separating system. The long-term operation demonstrated the high lipase stability affording less than 0.2% residual triglyceride in 22 batches. Therefore, the PBR system, which facilitates the separation of hydrophilic impurities, is applicable to the enzymatic biodiesel production from waste cooking oil.

Water activity dependence of performance of surface-displayed lipase in yeast cells: a unique water requirement for enzymatic synthetic reaction in organic media.

Water activity (a(w)) is a crucial parameter affecting enzymatic synthetic reactions in organic media. In this paper, we report on the a(w) dependence of surface-displayed lipases, genetically immobilized on yeast cells via fusion with cell wall proteins. When Saccharomyces cerevisiae displaying Rhizopus oryzae lipase was used for esterification in n-hexane, equilibrating the dried cells with water prior to the reaction markedly increased the reaction rate. An equilibration of the cells with various saturated salt solutions showed that the reaction rate increased with increasing a(w) of the salt solution, to give the best performance at a(w) of 1.0. Interestingly, this trend was extremely different from those of lipases in powder or resin-immobilized form. To determine whether the cell surface is responsible for the unique a(w) profiles, an investigation was carried out similarly using other lipase sources and yeast strains, which indicated that, in all the cells examined, a higher a(w) resulted in a higher reaction rate. Moreover, increasing a(w) was found to increase the cell surface hydrophobicity determined by an aqueous-hydrocarbon biphasic partitioning assay. These results indicate that lipases displayed on yeast cells show a unique a(w) dependence probably because of the variation in cell surface characteristics.

Process engineering and optimization of glycerol separation in a packed-bed reactor for enzymatic biodiesel production.

A process model for efficient glycerol separation during methanolysis in an enzymatic packed-bed reactor (PBR) was developed. A theoretical glycerol removal efficiency from the reaction mixture containing over 30% methyl esters was achieved at a high flow rate of 540 ml/h. To facilitate a stable operation of the PBR system, a batch reaction prior to continuous methanolysis was conducted using oils with different acid values and immobilized lipases pretreated with methyl esters. The reaction system successfully attained the methyl ester content of over 30% along with reduced viscosity and water content. Furthermore, to obtain a high methyl ester content above 96% continuously, long-term lipase stability was confirmed by operating a bench-scale PBR system for 550 h, in which the intermediates containing methyl esters and residual glycerides were fed into the enzyme-packed columns connected in series. Therefore, the developed process model is considered useful for industrial biodiesel production.

Activation of sphingomyelinase from Bacillus cereus by Zn2+ hitherto accepted as a strong inhibitor.

Sphingomyelinase (SMase) from Bacillus cereus has been known to be activated by Mg2+, Mn2+, and Co2+, but strongly inhibited by Zn2+. In the present study, we investigated the effects of several kinds of metal ions on the catalytic activity of B. cereus SMase, and found that the activity was inhibited by Zn2+ at its higher concentrations or at higher pH values, but unexpectedly activated at lower Zn2+ concentrations or at lower pH values. This result indicates that SMase possesses at least two different binding sites for Zn2+ and that the Zn2+ binding to the high-affinity site can activate the enzyme, whereas the Zn2+ binding to the low-affinity site can inactivate it. We also found that the binding of substrate to the enzyme was independent of the Zn2+ binding to the high-affinity site, but was competitively inhibited by the Zn2+ binding to the low-affinity site. The binding affinity of the metal ions to the site for activating the enzyme was determined to be in the rank-order of Mg2+ = Co2+ < Mn2+ < Zn2+. It was also demonstrated that these four metal ions competed with each other for the same binding site on the enzyme molecule.

Quantity and quality control of gastric proton pump in the endoplasmic reticulum by ubiquitin/proteasome system.

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the noncatalytic beta-subunit. These subunits are assembled in the endoplasmic reticulum (ER) and leave the ER to reach to the cell surface as a functional holoenzyme. We studied the quantity control mechanism of the H(+),K(+)-ATPase in the ER by using a heterologous expression system in human embryonic kidney 293 cells. The alpha-subunit in the alpha-expressing cells was degraded more rapidly than in the alpha+beta-expressing cells. It was stabilized, however, in the presence of a proteasome inhibitor, lactacystin. Polyubiquitination of the alpha-subunit was observed in the alpha-expressing cells as well as in the alpha+beta-expressing cells. The extent of polyubiquitination was higher in the former alpha-expressing cells especially in the presence of lactacystin. On the other hand, polyubiquitination of the beta-subunit was not observed in the absence and presence of lactacystin. When the alpha-subunit was coexpressed with a mutant beta-subunit that lacks alpha/beta assembly capacity, degradation of the alpha-subunit was accelerated in parallel with increased polyubiquitination of the alpha-subunit. These results indicate that the ubiquitin/proteasome system is involved in degradation of the unassembled alpha-subunits in the ER to control the cell surface expression of the functional alpha/beta holoenzymes.

Chromogenic assay for the activity of sphingomyelinase from Bacillus cereus and its application to the enzymatic hydrolysis of lysophospholipids.

We developed a convenient chromogenic assay method for the activity of sphingomyelinase (SMase) from Bacillus cereus. SMase reaction was quenched by Zn(2+), and the released phosphocholine was converted into a choline by the action of alkaline phosphatase. After that, the choline was converted into a chromogenic dye by the actions of choline oxidase and peroxidase in the presence of EDTA to trap the added Zn(2+) which could interfere with the choline oxidase/peroxidase reactions. Triton X-100 also was added to the reaction mixture, in order to remove turbidity generated from ceramide which had been produced by the SMase reaction. To test a large number of samples in a short period of time, this assay was performed using 96-well microtiter plates. This method proved to be applicable not only to the measurement of the hydrolysis of sphingomyelin but also to those of lysophosphatidylcholine (lysoPC) and lyso platelet-activating factor by B. cereus SMase. Using this method, the kinetic parameters (K(m) and k(cat)) for B. cereus SMase toward various types of substrates were then determined, and the effect of Triton X-100 on the hydrolysis of lysoPC was examined.

The cavity structure for docking the K(+)-competitive inhibitors in the gastric proton pump.

2-Methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080) is a reversible inhibitor specific for the gastric proton pump. The inhibition pattern is competitive with K(+). Here we studied the binding sites of this inhibitor on the putative three-dimensional structure of the gastric proton pump alpha-subunit that was constructed by homology modeling based on the structure of sarcoplasmic reticulum Ca(2+) pump. Alanine and serine mutants of Tyr(801) located in the fifth transmembrane segment of the gastric proton pump alpha-subunit retained the (86)Rb transport and K(+)-dependent ATPase (K(+)-ATPase) activities. These mutants showed 60-80-times lower sensitivity to SCH 28080 than the wild type in the (86)Rb transport activity. The K(+)-ATPase activities of these mutants were not completely inhibited by SCH 28080. The sensitivity to SCH 28080 was dependent on the bulkiness of the side chain at this position. Therefore, the side chain of Tyr(801) is important for the interaction with this inhibitor. In the three-dimensional structure of the E(2) form (conformation with high affinity for K(+)) of the gastric proton pump, Tyr(801) faces a cavity surrounded by the first, fourth, fifth, sixth, and eighth transmembrane segments and fifth/sixth, seventh/eighth, and ninth/tenth loops. SCH 28080 can dock in this cavity. However, SCH 28080 cannot dock in the same location in the E(1) form (conformation with high affinity for proton) of the gastric proton pump due to the drastic rearrangement of the transmembrane helices between the E(1) and E(2) forms. These results support the idea that this cavity is the binding pocket of SCH 28080.

Contribution of oligosaccharides to protection of the H,K-ATPase beta-subunit against trypsinolysis.

The proton-pumping H+,K+-adenosinetriphosphatase (H,K-ATPase), responsible for acid secretion by the gastric parietal cell, faces a harshly acidic environment, with some pepsin from neighboring chief cells, at its luminal surface. Its large catalytic alpha-subunit is mostly oriented cytoplasmically. The smaller beta-subunit (HKbeta), is mainly extracellular, with one transmembrane domain and a small cytoplasmic domain. Seven N-linked oligosaccharides in the extracellular domain of HKbeta are thought to contribute to protection of the H,K-ATPase, since previous work has shown that their complete removal, by peptide N-glycosidase F (PNGase F), greatly increased susceptibility of HKbeta to proteolysis. The possibility of graded protection by different numbers of oligosaccharides was investigated here with the use of mutant HKbeta cDNA, having various N-glycosylation sites mutated (Asn to Gln), transfected into HEK-293 cells. Membrane preparations, two days after transfection, were solubilized in 1% Triton X-100 and subjected to trypsinolysis (pH 8, 37 degrees C, trypsin:protein 1:10-1:25). Relative amounts of HKbeta remaining after 20 min trypsin were determined, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probing of Western blots with an antibody to the HKbeta extracellular domain, by chemiluminescent development of blots and densitometry of resulting films. Maturely glycosylated HKbeta was made significantly more susceptible to trypsin than wild type when at least five oligosaccharides were deleted, while the high-mannose form (pre-beta), from the endoplasmic reticulum, became significantly more susceptible than wild-type pre-beta with removal of only two or more oligosaccharides. For each mutant, and wild type, pre-beta was consistently more susceptible than the mature form. While the number, and kind, of oligosaccharides seem to affect protection for HKbeta against trypsinolysis, other aspects of protein maturation, including proper folding of peptide domains and possible subtle alterations of conformation during Golgi processing, are also likely to contribute to this protection.