N-Glycosylation Is Required for Matriptase-2 Autoactivation and Ectodomain Shedding

Matriptase-2 is a hepatic membrane serine protease that regulates iron homeostasis. Defects in matriptase-2 cause iron deficiency anemia. In cells, matriptase-2 is synthesized as a zymogen. To date, how matriptase-2 expression and activation are regulated remains poorly understood. Here we expressed human matriptase-2 in HEK293 and hepatic BEL-7402, SMMC-7721, and QGY-7703 cells. By labeling cell surface proteins and Western analysis, we examined matriptase-2 cell surface expression, zymogen activation, and ectodomain shedding. Our results show that matriptase-2 was activated on the cell surface but not intracellularly. Activated matriptase-2 underwent ectodomain shedding, producing soluble fragments in the conditioned medium. By testing inactive mutants, R576A and S762A, we found that matriptase-2 activation and shedding were mediated by its own catalytic activity and that the one-chain form of matriptase-2 had little activity in ectodomain shedding. We made additional matriptase-2 mutants, N136Q, N184Q, N216Q, N338Q, N433Q, N453Q, and N518Q, in which each of the predicted N-glycosylation sites was mutated. All of these mutants were expressed on the cell surface. However, mutants N216Q, N453Q, and N518Q, but not the other mutants, had impaired zymogen activation and ectodomain shedding. Our results indicate that N-glycans at specific sites are critical for matriptase-2 activation. Together, these data provide new insights into the cell surface expression, zymogen activation, and ectodomain shedding of matriptase-2.     

Ectodomain shedding and autocleavage of the cardiac membrane protease corin

Corin is a cardiac membrane protease that activates natriuretic peptides. It is unknown how corin function is regulated. Recently, soluble corin was detected in human plasma, suggesting that corin may be shed from cardiomyocytes. Here we examined soluble corin production and activity and determined the proteolytic enzymes responsible for corin cleavage. We expressed human corin in HEK 293 cells and detected three soluble fragments of ～180, ～160, and ～100 kDa, respectively, in the cultured medium by Western blot analysis. All three fragments were derived from activated corin molecules. Similar results were obtained in HL-1 cardiomyocytes. Using protease inhibitors, ionomycin and phorbol myristate acetate stimulation, small interfering RNA knockdown, and site-directed mutagenesis, we found that ADAM10 was primarily responsible for shedding corin in its juxtamembrane region to release the ～180-kDa fragment, corresponding to the near-entire extracellular region. In contrast, the ～160- and ～100-kDa fragments were from corin autocleavage at Arg-164 in frizzled 1 domain and Arg-427 in LDL receptor 5 domain, respectively. In functional studies, the ～180-kDa fragment activated atrial natriuretic peptide, whereas the ～160- and ～100-kDa fragments did not. Our data indicate that ADAM-mediated shedding and corin autocleavage are important mechanisms regulating corin function and preventing excessive, potentially hazardous, proteolytic activities in the heart.     

Proteolysis-induced N-terminal Ectodomain Shedding of the Integral Membrane Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Is Accompanied by Tyrosine Phosphorylation of Its C-terminal Domain and Recruitment of Src and PKCδ

CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKCδ to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein.     

Roles for Trafficking and O-Linked Glycosylation in the Turnover of Model Cell Surface Proteins

Proteins targeted to the plasma membrane (PM) of cells are degraded at different rates. Sorting motifs contained within the cytoplasmic domains of transmembrane proteins, post-translational modifications (e.g. ubiquitination), and assembly into multiprotein or protein-lipid complexes all may affect the efficiency of endocytosis and recycling and influence the delivery to degradative compartments. Using the SNAP-tag labeling system, we examined the turnover of a model PM protein, the α chain of the interleukin-2 receptor (Tac). The surface lifetimes of SNAP-Tac fusions were influenced by their mode of entry into cells (clathrin-dependent versus clathrin-independent), their orientation in the PM (transmembrane versus glycosylphosphatidylinositol-anchored), and ubiquitination in their cytosolic domains. In addition, shedding of SNAP-Tac into the medium was greatly influenced by its O-linked glycosylation status. For a number of PM proteins, delivery to lysosomes and ectodomain shedding represent distinct parallel mechanisms to determine protein half-life.     

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O_δ2–C_γ bond appears to be a double bond, with O_δ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O_δ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF_obs − DF_calc density above 2.5 σ next to O_δ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK_a of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK_a histidine theory.     

Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P₁ (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P′₂ favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P₁ and P′₂ substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin·APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.     

The N- and C-terminal Domains of Tomosyn Play Distinct Roles in Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Binding and Fusion Regulation

Tomosyn negatively regulates SNARE-dependent exocytic pathways including insulin secretion, GLUT4 exocytosis, and neurotransmitter release. The molecular mechanism of tomosyn, however, has not been fully elucidated. Here, we reconstituted SNARE-dependent fusion reactions in vitro to recapitulate the tomosyn-regulated exocytic pathways. We then expressed and purified active full-length tomosyn and examined how it regulates the reconstituted SNARE-dependent fusion reactions. Using these defined fusion assays, we demonstrated that tomosyn negatively regulates SNARE-mediated membrane fusion by inhibiting the assembly of the ternary SNARE complex. Tomosyn recognizes the t-SNARE complex and prevents its pairing with the v-SNARE, therefore arresting the fusion reaction at a pre-docking stage. The inhibitory function of tomosyn is mediated by its C-terminal domain (CTD) that contains an R-SNARE-like motif, confirming previous studies carried out using truncated tomosyn fragments. Interestingly, the N-terminal domain (NTD) of tomosyn is critical (but not sufficient) to the binding of tomosyn to the syntaxin monomer, indicating that full-length tomosyn possesses unique features not found in the widely studied CTD fragment. Finally, we showed that the inhibitory function of tomosyn is dominant over the stimulatory activity of the Sec1/Munc18 protein in fusion. We suggest that tomosyn uses its CTD to arrest SNARE-dependent fusion reactions, whereas its NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction.     

Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites

Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK¹⁷⁸), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.     

Direct Observation and Quantitative Analysis of Lck Exchange between Plasma Membrane and Cytosol in Living T Cells

Palmitoylation represents a common motif for anchorage of cytosolic proteins to the plasma membrane. Being reversible, it allows for controlled exchange between cytosolic and plasma membrane-bound subpopulations. In this study, we present a live cell single molecule approach for quantifying the exchange kinetics of plasma membrane and cytosolic populations of fluorescently labeled Lck, the key Src family kinase involved in early T cell signaling. Total internal reflection (TIR) fluorescence microscopy was employed for confining the analysis to membrane-proximal molecules. Upon photobleaching Lck-YFP in TIR configuration, fluorescence recovery proceeds first via the cytosol outside of the evanescent field, so that in the early phase fluorescence signal arises predominantly from membrane-proximal cytosolic Lck. The diffusion constant of each molecule allowed us to distinguish whether the molecule has already associated with the plasma membrane or was still freely diffusing in the cytosol. From the number of molecules that inserted during the recovery time we quantified the insertion kinetics: on average, membrane-proximal molecules within the evanescent field needed ∼400 ms to be inserted. The average lifetime of Lck in the plasma membrane was estimated at 50 s; together with the mobility of 0.26 μm²/s this provides sufficient time to explore the surface of the whole T cell before dissociation into the cytosol. Experiments on palmitoylation-deficient Lck mutants yielded similar on-rates, but substantially increased off-rates. We discuss our findings based on a model for the plasma membrane association and dissociation kinetics of Lck, which accounts for reversible palmitoylation on cysteine 3 and 5.     

Distinct Regulation of Cytoplasmic Calcium Signals and Cell Death Pathways by Different Plasma Membrane Calcium ATPase Isoforms in MDA-MB-231 Breast Cancer Cells

Plasma membrane calcium ATPases (PMCAs) actively extrude Ca(2+) from the cell and are essential components in maintaining intracellular Ca(2+) homeostasis. There are four PMCA isoforms (PMCA1-4), and alternative splicing of the PMCA genes creates a suite of calcium efflux pumps. The role of these different PMCA isoforms in the control of calcium-regulated cell death pathways and the significance of the expression of multiple isoforms of PMCA in the same cell type are not well understood. In these studies, we assessed the impact of PMCA1 and PMCA4 silencing on cytoplasmic free Ca(2+) signals and cell viability in MDA-MB-231 breast cancer cells. The PMCA1 isoform was the predominant regulator of global Ca(2+) signals in MDA-MB-231 cells. PMCA4 played only a minor role in the regulation of bulk cytosolic Ca(2+), which was more evident at higher Ca(2+) loads. Although PMCA1 or PMCA4 knockdown alone had no effect on MDA-MB-231 cell viability, silencing of these isoforms had distinct consequences on caspase-independent (ionomycin) and -dependent (ABT-263) cell death. PMCA1 knockdown augmented necrosis mediated by the Ca(2+) ionophore ionomycin, whereas apoptosis mediated by the Bcl-2 inhibitor ABT-263 was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4 silencing in promoting ABT-263-induced cell death. This study demonstrates distinct roles for PMCA1 and PMCA4 in the regulation of calcium signaling and cell death pathways despite the widespread distribution of these two isoforms. The targeting of some PMCA isoforms may enhance the effectiveness of therapies that act through the promotion of cell death pathways in cancer cells.     

Distinct Roles of the C-terminal 11th Transmembrane Helix and Luminal Extension in the Partial Reactions Determining the High Ca2+ Affinity of Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2b (SERCA2b)

The molecular mechanism underlying the characteristic high apparent Ca²⁺ affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11-amino acid luminal extension (LE). The effects of each of these parts and their interactions with the SERCA environment were examined by transient kinetic analysis of the partial reaction steps in the Ca²⁺ transport cycle in mutant and chimeric Ca²⁺-ATPase constructs. Manipulations to the LE of SERCA2b markedly increased the rate of Ca²⁺ dissociation from Ca₂E1. Addition of the SERCA2b tail to SERCA1a slowed Ca²⁺ dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca₂E1. The interaction of LE with L7/8 is also important for the low rate of the Ca₂E1P → E2P conformational transition. These findings can be rationalized in terms of stabilization of the Ca₂E1 and Ca₂E1P forms by docking of the LE near L7/8. By contrast, low rates of E2P dephosphorylation and E2 → E1 transition in SERCA2b depend critically on TM11, particularly in a SERCA2 environment, but do not at all depend on the LE or L7/8. This indicates that interaction of TM11 with SERCA2-specific sequence element(s) elsewhere in the structure is critical in the Ca²⁺-free E2/E2P states. Collectively these properties ensure a higher Ca²⁺ affinity of SERCA2b relative to other SERCA isoforms, not only on the cytosolic side, but also on the luminal side.     

苦瓜果实不同发育时期细胞壁组分及相关酶活性的差异分析

为了解苦瓜(Momordica charantia)果实品质差异的原因，以厚肉型种质‘LX1-3’和薄肉型种质‘ZK54’为材料，对果实发育过程中细胞壁组分含量及相关酶活性进行了分析。结果表明，花后17 d，‘LX1-3’果实的横径(FD)、腔径(FLD)、果肉厚度(PT)、单瓜鲜质量(FFW)和干质量(FDW)均超过‘ZK54’。细胞壁组分和酶活性表现品种间差异，水溶性果胶含量整体水平表现为厚肉型高于薄肉型，且与PT、FFW和FDW呈显著正相关；花后17~23 d ‘LX1-3’的半纤维素(HCE)和纤维素(CE)含量均高于‘ZK54’；花后3 d，两种质的β-半乳糖苷酶(β-Gal)和β-木糖苷酶(β-Xyl)活性显著高于其他3个时期，多聚半乳糖醛酸酶(PG)、β-Gal和果胶酶变化趋势与离子型果胶和共价型果胶含量的变化一致。β-Gal、β-Xyl和纤维素酶活性与5个生长性状间呈极显著/显著负相关，PG与FD、FLD和PT呈极显著/显著负相关。因此，细胞壁组分和酶活性与果实发育密切相关，β-Xyl和β-Gal在苦瓜早期发育中发挥主要效应，而HCE和CE对果实中后期发育影响较大。