Functional linkage between the endoplasmic reticulum protein Hsp47 and procollagen expression in human vascular smooth muscle cells

Hsp47 is a heat stress protein that interacts with procollagen in the lumen of the endoplasmic reticulum, which is vital for collagen elaboration and embryonic viability. The precise actions of Hsp47 remain unclear, however. To evaluate the effects of Hsp47 on collagen production we infected human vascular smooth muscle cells (SMCs) with a retrovirus containing Hsp47 cDNA. SMCs overexpressing Hsp47 secreted type I procollagen faster than SMCs transduced with empty vector, yielding a greater accumulation of pro alpha1(I) collagen in the extracellular milieu. Interestingly, the amount of intracellular pro alpha1(I) collagen was also increased. This was associated with an unexpected increase in the rate of pro alpha1(I) collagen chain synthesis and 2.5-fold increase in pro alpha1(I) collagen mRNA expression, without a change in fibronectin expression. This amplification of procollagen expression, synthesis, and secretion by Hsp47 imparted SMCs with an enhanced capacity to elaborate a fibrillar collagen network. The effects of Hsp47 were qualitatively distinct from, and independent of, those of ascorbate and the combination of both factors yielded an even more intricate fibril network. Given the in vitro impact of altered Hsp47 expression on procollagen production, we sought evidence for interindividual variability in Hsp47 expression and identified a common, single nucleotide polymorphism in the Hsp47 gene promoter among African Americans that significantly reduced promoter activity. Together, these findings indicate a novel means by which type I collagen production is regulated by the endoplasmic reticulum constituent, Hsp47, and suggest a potential basis for inherent differences in collagen production within the population.     

The globular domain of the proalpha 1(I) N-propeptide is not required for secretion, processing by procollagen N-proteinase, or fibrillogenesis of type I collagen in mice.

The globular domain in the NH(2)-terminal propeptide (N-propeptide) of the proalpha1(I) chain is largely encoded by exon 2 of the Col1a1 gene and has been implicated in a number of processes that are involved in the biogenesis, maturation, and function of type I collagen. These include intracellular chain association, transcellular transport and secretion, proteolytic processing of the precursor, feedback regulation of synthesis, and control of fibrillogenesis. However, none of these proposed functions has been firmly established. To evaluate the function of this procollagen domain we have used a targeted mutagenesis approach to generate mice that lack exon 2 in the Col1a1 gene. Mouse lines were established on both a mixed 129 OlaHsd/Sv and C57BL/6 background and a pure 129 OlaHsd/Sv background. Adult mice on the mixed background are normal in appearance and are fertile. To the extent that they have been studied, procollagen synthesis, secretion, and proteolytic processing are normal in these mice, and collagen fibrillogenesis is only slightly altered. However, breeding of heterozygous mutant mice on the 129 background generated homozygous mutants at only 64% of the expected frequency. These findings suggest that although the N-propeptide is not essential for collagen biogenesis in mice it may play some essential role during embryonic development.     

SPARC regulates processing of procollagen I and collagen fibrillogenesis in dermal fibroblasts

A characterization of the factors that control collagen fibril formation is critical for an understanding of tissue organization and the mechanisms that lead to fibrosis. SPARC (secreted protein acidic and rich in cysteine) is a counter-adhesive protein that binds collagens. Herein we show that collagen fibrils in SPARC-null skin from mice 1 month of age were inefficient in fibril aggregation and accumulated in the diameter range of 60-70 nm, a proposed intermediate in collagen fibril growth. In vitro, procollagen I produced by SPARC-null dermal fibroblasts demonstrated an initial preferential association with cell layers, in comparison to that produced by wild-type fibroblasts. However, the collagen I produced by SPARC-null cells was not efficiently incorporated into detergent-insoluble fractions. Coincident with an initial increase in cell association, greater amounts of total collagen I were present as processed forms in SPARC-null versus wild-type cells. Addition of recombinant SPARC reversed collagen I association with cell layers and decreased the processing of procollagen I in SPARC-null cells. Although collagen fibers formed on the surface of SPARC-null fibroblasts earlier than those on wild-type cells, fibers on SPARC-null fibroblasts did not persist. We conclude that SPARC mediates the association of procollagen I with cells, as well as its processing and incorporation into the extracellular matrix.     

Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum

The 47,000-D collagen-binding glycoprotein, heat shock protein 47 (HSP47), is a stress-inducible protein localized in the ER of collagen- secreting cells. The location and collagen-binding activity of this protein led to speculation that HSP47 might participate in collagen processing. Chemical crosslinking studies were used to test this hypothesis both before and after the perturbation of procollagen processing. The association of procollagen with HSP47 was demonstrated using cleavable bifunctional crosslinking reagents. HSP47 and procollagen were shown to be coprecipitated by the treatment of intact cells with anti-HSP47 or with anticollagen antibodies. Furthermore, several proteins residing in the ER were noted to be crosslinked to and coprecipitated with HSP47, suggesting that these ER-resident proteins may form a large complex in the ER. When cells were heat shocked, or when stable triple-helix formation was inhibited by treatment with alpha,alpha'-dipyridyl, coprecipitation of procollagen with HSP47 was increased. This increase was due to the inhibition of procollagen secretion and to the accumulation of procollagen in the ER. Pulse label and chase experiments revealed that coprecipitated procollagen was detectable as long as procollagen was present in the endoplasmic reticulum of alpha,alpha'-dipyridyl-treated cells. Under normal growth conditions, coprecipitated procollagen was observed to decrease after a chase period of 10-15 min, whereas total procollagen decreased only after 20-25 min. In addition, the intracellular association between HSP47 and procollagen was shown to be disrupted by a change in physiological pH, suggesting that the dissociation of procollagen from HSP47 is pH dependent. These findings support a specific role for HSP47 in the intracellular processing of procollagen, and provide evidence of a new category of "molecular chaperones" in terms of its substrate specificity and the dissociation mechanism.     

Insufficient folding of type IV collagen and formation of abnormal basement membrane-like structure in embryoid bodies derived from Hsp47-null embryonic stem cells

Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47-/- embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47-/- embryoid bodies were often disorganized, a result that suggested abnormal function of the basement membrane under the visceral endoderm. Rate of type IV collagen secretion by Hsp47-/- cells was fourfold lower than that of Hsp47+/+ cells. Furthermore, type IV collagen secreted from Hsp47-/- cells was much more sensitive to protease digestion than was type IV collagen secreted from Hsp47+/+ cells, which suggested insufficient or incorrect triple helix formation in type IV collagen in the absence of Hsp47. These results indicate for the first time that Hsp47 is required for the molecular maturation of type IV collagen and suggest that misfolded type IV collagen causes abnormal morphology of embryoid bodies.     

Reciprocal relationship between alpha1,2 mannosidase processing and reglucosylation in the rough endoplasmic reticulum of Man-P-Dol deficient cells.

The study of the glycosylation pathway of a mannosylphosphoryldolichol-deficient CHO mutant cell line (B3F7) reveals that truncated Glc(0-3)Man5GlcNAc2 oligosaccharides are transferred onto nascent proteins. Pulse-chase experiments indicate that these newly synthesized glycoproteins are retained in intracellular compartments and converted to Man4GlcNAc2 species. In this paper, we demonstrate that the alpha1,2 mannosidase, which is involved in the processing of Man5GlcNAc2 into Man4GlcNAc2, is located in the rough endoplasmic reticulum. The enzyme was shown to be inhibited by kifunensine and deoxymannojirimycin, indicating that it is a class I mannosidase. In addition, Man4GlcNAc2 species were produced at the expense of Glc1Man5GlcNAc2 species. Thus, the trimming of Man5GlcNAc2 to Man4GlcNAc2, which is catalyzed by this mannosidase, could be involved in the control of the glucose-dependent folding pathway.     

Type I collagen fibrillogenesis: initiation via reversible linear and lateral growth steps

Type I collagen fibrillogenesis in vitro has been studied by laser light scattering, and the results indicate that initiation of aggregation involves at least two steps. Step I of aggregation involves no change in the intensity of scattered light at an angle of 90° and is accompanied by a decrease in the diffusion coefficient. Step II is characterized by an increased intensity of scattered light and decreased diffusion coefficients. Theoretical calculations using the Stokes-Einstein equation for the translational diffusion coefficient and the Perrin equation for the frictional coefficient of a prolate ellipsoid indicate that the step I aggregates are 4D staggered linear dimers and trimers 570 and 845 nm long, whereas step II aggregates are greater than 950 nm in length. These dimensions are similar to those previously reported based on physicochemical measurements and electron microscopy. It is proposed that the rate and extent of fibrillogenesis in vitro is controlled by the concentration of the linear aggregates and that the effects of temperature and collagen concentration on fibrillogenesis previously observed are qualitatively explained in terms of their effects on the concentration of these aggregates.     

Biosynthesis and processing of ribophorins in the endoplasmic reticulum

Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.     

Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation

The search for novel and more efficient chemo-agents against malignant osteoblastoma is important. In this study, we examined the potential anti-osteoblastoma function of bufotalin, and studied the underlying mechanisms. Our results showed that bufotalin induced osteoblastoma cell death and apoptosis in dose- and time-dependent manners. Further, bufotalin induced endoplasmic reticulum (ER) stress activation in osteoblastoma cells, the latter was detected by the induction of C/EBP homologous protein (CHOP), phosphorylation of inositol-requiring enzyme 1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), as well as caspase-12 activation. Conversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP depletion by shRNA significantly inhibited bufotalin-induced osteoblastoma cell death and apoptosis. Finally, by using a mice xenograft model, we demonstrated that bufotalin inhibited U2OS osteoblastoma cell growth in vivo. In summary, our results suggest that ER stress contributes to bufotalin-induced apoptosis in osteoblastoma cells. Bufotalin might be investigated as a novel anti-osteoblastoma agent.     

Golgi inheritance in mammalian cells is mediated through endoplasmic reticulum export activities

Golgi inheritance during mammalian cell division occurs through the disassembly, partitioning, and reassembly of Golgi membranes. The mechanisms responsible for these processes are poorly understood. To address these mechanisms, we have examined the identity and dynamics of Golgi proteins within mitotic membranes using live cell imaging and electron microscopy techniques. Mitotic Golgi fragments, seen in prometaphase and telophase, were found to localize adjacent to endoplasmic reticulum (ER) export domains, and resident Golgi transmembrane proteins cycled rapidly into and out of these fragments. Golgi proteins within mitotic Golgi haze-seen during metaphase-were found to redistribute with ER markers into fragments when the ER was fragmented by ionomycin treatment. The temperature-sensitive misfolding mutant ts045VSVG protein, when localized to the Golgi at the start of mitosis, became trapped in the ER at the end of mitosis in cells shifted to 40 degrees C. Finally, reporters for Arf1 and Sar1 activity revealed that Arf1 and Sar1 undergo sequential inactivation during mitotic Golgi breakdown and sequential reactivation upon Golgi reassembly at the end of mitosis. Together, these findings support a model of mitotic Golgi inheritance that involves inhibition and subsequent reactivation of cellular activities controlling the cycling of Golgi components into and out of the ER.     

TRAM2 protein interacts with endoplasmic reticulum Ca2+ pump Serca2b and is necessary for collagen type I synthesis

Cotranslational insertion of type I collagen chains into the lumen of the endoplasmic reticulum (ER) and their subsequent folding into a heterotrimeric helix is a complex process which requires coordinated action of the translation machinery, components of translocons, molecular chaperones, and modifying enzymes. Here we describe a role for the protein TRAM2 in collagen type I expression in hepatic stellate cells (HSCs) and fibroblasts. Activated HSCs are collagen-producing cells in the fibrotic liver. Quiescent HSCs produce trace amounts of type I collagen, while upon activation collagen synthesis increases 50- to 70-fold. Likewise, expression of TRAM2 dramatically increases in activated HSCs. TRAM2 shares 53% amino acid identity with the protein TRAM, which is a component of the translocon. However, TRAM2 has a C terminus with only a 15% identity. The C-terminal part of TRAM2 interacts with the Ca(2+) pump of the ER, SERCA2b, as demonstrated in a Saccharomyces cerevisiae two-hybrid screen and by immunoprecipitations in human cells. TRAM2 also coprecipitates with anticollagen antibody, suggesting that these two proteins interact. Deletion of the C-terminal part of TRAM2 inhibits type I collagen synthesis during activation of HSCs. The pharmacological inhibitor of SERCA2b, thapsigargin, has a similar effect. Depletion of ER Ca(2+) with thapsigargin results in inhibition of triple helical collagen folding and increased intracellular degradation. We propose that TRAM2, as a part of the translocon, is required for the biosynthesis of type I collagen by coupling the activity of SERCA2b with the activity of the translocon. This coupling may increase the local Ca(2+) concentration at the site of collagen synthesis, and a high Ca(2+) concentration may be necessary for the function of molecular chaperones involved in collagen folding.     

Nonapoptotic death of Saccharomyces cerevisiae cells that is stimulated by Hsp90 and inhibited by calcineurin and Cmk2 in response to endoplasmic reticulum stresses

Endoplasmic reticulum (ER) stress can trigger apoptosis and necrosis in many types of mammalian cells. Previous studies in yeast found little or no cell death in response to the ER stressor tunicamycin, but a recent study suggested widespread apoptosis-like death. Here we show that wild-type laboratory Saccharomyces cerevisiae cells responding to tunicamycin die by nonapoptotic mechanisms in low-osmolyte culture media and survive for long periods of time in standard synthetic media. Survival requires calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase, but none of its known targets. The Ca(2+)/calmodulin-dependent protein kinase Cmk2 was identified as an indirect target of calcineurin that suppresses death of calcineurin-deficient cells. Death of Cmk2- and/or calcineurin-deficient S. cerevisiae cells was preceded by accumulation of reactive oxygen species but was not associated with hallmarks of apoptosis and was not dependent on Mca1, Aif1, Nuc1, or other factors implicated in apoptosis-like death. Cmk2 and calcineurin also independently suppressed the death of S. cerevisiae cells responding to dithiothreitol or miconazole, a common azole-class antifungal drug. Though inhibitors of Hsp90 have been shown to diminish calcineurin signaling in S. cerevisiae and to synergistically inhibit growth in combination with azoles, they did not stimulate death of S. cerevisiae cells in combination with miconazole or tunicamycin, and instead they prevented the death of calcineurin- and Cmk2-deficient cells. These findings reveal a novel prodeath role for Hsp90 and antideath roles for calcineurin and Cmk2 that extend the life span of S. cerevisiae cells responding to both natural and clinical antifungal compounds.     

Syntaxin 17 is abundant in steroidogenic cells and implicated in smooth endoplasmic reticulum membrane dynamics

The endoplasmic reticulum (ER) consists of subcompartments that have distinct protein constituents, morphological appearances, and functions. To understand the mechanisms that regulate the intricate and dynamic organization of the endoplasmic reticulum, it is important to identify and characterize the molecular machinery involved in the assembly and maintenance of the different subcompartments. Here we report that syntaxin 17 is abundantly expressed in steroidogenic cell types and specifically localizes to smooth membranes of the ER. By immunoprecipitation analyses, syntaxin 17 exists in complexes with a syntaxin regulatory protein, rsly1, and/or two intermediate compartment SNARE proteins, rsec22b and rbet1. Furthermore, we found that syntaxin 17 is anchored to the smooth endoplasmic reticulum through an unusual mechanism, requiring two adjacent hydrophobic domains near its carboxyl terminus. Converging lines of evidence indicate that syntaxin 17 functions in a vesicle-trafficking step to the smooth-surfaced tubular ER membranes that are abundant in steroidogenic cells.     

Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen

As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.     

Reduced endoplasmic reticulum luminal calcium links saturated fatty acid-mediated endoplasmic reticulum stress and cell death in liver cells

Chronic exposure to elevated free fatty acids, in particular long chain saturated fatty acids, provokes endoplasmic reticulum (ER) stress and cell death in a number of cell types. The perturbations to the ER that instigate ER stress and activation of the unfolded protein in response to fatty acids in hepatocytes have not been identified. The present study employed H4IIE liver cells and primary rat hepatocytes to examine the hypothesis that saturated fatty acids induce ER stress via effects on ER luminal calcium stores. Exposure of H4IIE liver cells and primary hepatocytes to palmitate and stearate reduced thapsigargin-sensitive calcium stores and increased biochemical markers of ER stress over similar time courses (6 h). These changes preceded cell death, which was only observed at later time points (16 h). Co-incubation with oleate prevented the reduction in calcium stores, induction of ER stress markers and cell death observed in response to palmitate. Inclusion of calcium chelators, BAPTA-AM or EGTA, reduced palmitate- and stearate-mediated enrichment of cytochrome c in post-mitochondrial supernatant fractions and cell death. These data suggest that redistribution of ER luminal calcium contributes to long chain saturated fatty acid-mediated ER stress and cell death.     

Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum

Hsp70 molecular chaperones and their co-chaperones work together in various cellular compartments to guide the folding of proteins and to aid the translocation of proteins across membranes. Hsp70s stimulate protein folding by binding exposed hydrophobic sequences thereby preventing irreversible aggregation. Hsp40s stimulate the ATPase activity of Hsp70s and target unfolded proteins to Hsp70s. Genetic and biochemical evidence supports a role for cytosolic Hsp70s and Hsp40s in the post-translational translocation of precursor proteins into endoplasmic reticulum and mitochondria. To gain mechanistic insight, we measured the effects of Saccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p (Hsp40) on the translocation of histidine-tagged prepro-alpha-factor (ppalphaF6H) into microsomes. Radiolabeled ppalphaF6H was affinity purified from wheat germ translation reactions (or Escherichia coli) to remove endogenous chaperones. We demonstrated that either Ssa1p or Ydj1p stimulates post-translational translocation by preventing ppalphaF6H aggregation. The binding and/or hydrolysis of ATP by Ssa1p were required to maintain the translocation competence of ppalphaF6H. To clarify the contributions of membrane-bound and cytosolic Ydj1p, we compared the efficiency of chaperone-dependent translocation into wild-type and Ydj1p-deficient microsomes. Neither soluble nor membrane-bound Ydj1p was essential for post-translational protein translocation. The ability of Ssa1p, Ydj1p, or both chaperones to restore the translocation competence of aggregated ppalphaF6H was negligible.     

Hepatitis B virus MHBs antigen is selectively sensitive to glucosidase-mediated processing in the endoplasmic reticulum.

Previous studies have shown that hepatitis B virus (HBV) secretion from HepG 2.2.15 cells is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase under conditions where secretion of cellular glycoproteins are not detectably affected. The 2.2.15 cells are derived from HepG2 and contain intact dimers of the viral genome. They produce and secrete infectious HBV. The secretion of the viral envelope polypeptide, MHBs, was selectively and quantitatively reduced from 2.2.15 cells in which glucosidase was inhibited, whereas the envelope polypeptide, SHBs, was relatively insensitive, being as resistant as were most host glycoproteins. Because 2.2.15 cells express all HBV ORFs, it seemed possible that the sensitivity of MHBs secretion involved its interaction with the viral nucleocapsid or other viral gene products. The work reported here showed that MHBs secretion from HepG2 cells transfected with a plasmid that expresses only the MHBs polypeptide was as sensitive to glucosidase inhibitors as it was from 2.2.15 cells. These data show that the sensitivity of the MHBs polypeptide secretion to glucosidase inhibitors is entirely encrypted within its structural gene. The reasons the MHBs polypeptide, but not SHBs, is so sensitive to glucosidase processing are discussed.     

Expression and localization of collagen-binding stress protein Hsp47 in mouse embryo development: comparison with types I and II collagen

Heat shock protein (Hsp)47 is a collagen-binding stress protein localized in the endoplasmic reticulum and is thought to have chaperone-like functions that are specific to procollagen biosynthesis. In previous papers, we reported that the expression of Hsp47 is closely correlated with that of various types of collagen in various cell lines and also in the progression of experimental liver fibrosis. In the present study, the expression of Hsp47 was examined during the development of mouse embryos by immunostaining with an anti-Hsp47 antiserum. The spatio-temporal correlation of the expression of Hsp47 with those of types I and II collagen was also examined using specific antisera. Hsp47 expression during embryogenesis was observed mainly in mesoderm and in tissues that are derived from mesoderm, such as connective tissue, cartilage, bone, notochord and somites. Hsp47 was also detected in tissues derived from the neural crest mesenchyme. In the central nervous system, Hsp47 was detected in some restricted regions where cells proliferate, such as the ventral area of the neural tube and choroid plexus. Immunostaining for types I and II collagen revealed the spatial and temporal correlations of the expression of these proteins with that of Hsp47. These results suggest the biological importance of Hsp47 as a collagen-specific molecular chaperone in the mouse developmental program.     

Crystal structure of a class I alpha1,2-mannosidase involved in N-glycan processing and endoplasmic reticulum quality control

Mannose trimming is not only essential for N-glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins. The crystal structure of the class I alpha1, 2-mannosidase that trims Man(9)GlcNAc(2) to Man(8)GlcNAc(2 )isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (alphaalpha)(7)-barrel in which an N-glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion. The observed protein-carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases.