Spatial position is a key determinant of N-glycan functionality of the scavenger receptor cysteine-rich domain of human hepsin.

The scavenger receptor cysteine-rich (SRCR) domain is a key constituent in diverse proteins. N-glycosylation is important in protein expression and function. In the SRCR domain of different proteins, N-glycosylation sites and functionality vary substantially. In this study, we examined the importance of N-glycosylation site positions in the SRCR domain of hepsin, a type II transmembrane serine protease involved in many pathophysiological processes. We analysed hepsin mutants with alternative N-glycosylation sites in the SRCR and protease domains using three-dimensional modelling, site-directed mutagenesis, HepG2 cell expression, immunostaining, and western blotting. We found that the N-glycan function in the SRCR domain in promoting hepsin expression and activation on the cell surface cannot be replaced by alternatively created N-glycans in the protease domain. Within the SRCR domain, the presence of an N-glycan in a confined surface area was essential for calnexin-assisted protein folding, endoplasmic reticulum (ER) exiting, and zymogen activation of hepsin on the cell surface. Hepsin mutants with alternative N-glycosylation sites on the opposite side of the SRCR domain were trapped by ER chaperones, resulting in the activation of the unfolded protein response in HepG2 cells. These results indicate that the spatial N-glycan positioning in the SRCR domain is a key determinant in the interaction with calnexin and subsequent cell surface expression of hepsin. These findings may help to understand the conservation and functionality of N-glycosylation sites in the SRCR domains of different proteins.

Transmembrane serine protease TMPRSS2 implicated in SARS-CoV-2 infection is autoactivated intracellularly and requires N-glycosylation for regulation.

Transmembrane protease serine 2 (TMPRSS2) is a membrane-bound protease expressed in many human epithelial tissues, including the airway and lung. TMPRSS2-mediated cleavage of viral spike protein is a key mechanism in severe acute respiratory syndrome coronavirus 2 activation and host cell entry. To date, the cellular mechanisms that regulate TMPRSS2 activity and cell surface expression are not fully characterized. In this study, we examined two major post-translational events, zymogen activation and N-glycosylation, in human TMPRSS2. In experiments with human embryonic kidney 293, bronchial epithelial 16HBE, and lung alveolar epithelial A549 cells, we found that TMPRSS2 was activated via intracellular autocatalysis and that this process was blocked in the presence of hepatocyte growth factor activator inhibitors 1 and 2. By glycosidase digestion and site-directed mutagenesis, we showed that human TMPRSS2 was N-glycosylated. N-glycosylation at an evolutionarily conserved site in the scavenger receptor cysteine-rich domain was required for calnexin-assisted protein folding in the endoplasmic reticulum and subsequent intracellular trafficking, zymogen activation, and cell surface expression. Moreover, we showed that TMPRSS2 cleaved severe acute respiratory syndrome coronavirus 2 spike protein intracellularly in human embryonic kidney 293 cells. These results provide new insights into the cellular mechanism in regulating TMPRSS2 biosynthesis and function. Our findings should help to understand the role of TMPRSS2 in major respiratory viral diseases.

CD320 expression and apical membrane targeting in renal and intestinal epithelial cells.

Vitamin B12 is an essential nutrient acquired via dietary intake. Receptor-mediated endocytosis is a key mechanism in vitamin B12 absorption, cellular uptake, and reabsorption. CD320 is a type I transmembrane protein responsible for cellular uptake of vitamin B12 in peripheral tissues. In this study, we examined segmental distribution and cellular expression of CD320 in mouse kidneys and intestines. We show that CD320 is expressed on the luminal surface in the small intestine and in proximal tubules in the kidney, suggesting that, in addition to its role in vitamin B12 uptake in peripheral tissues, CD320 may participate in vitamin B12 absorption in the small intestine and reabsorption in the kidney. Moreover, we show that an amino acid motif, DSSDE, in the second low-density lipoprotein receptor class A domain of CD320 is a key apical membrane targeting signal in both renal and intestinal epithelial cells. Mutations or deletion of this motif abolish the specific apical membrane expression of CD320 in polarized Madin-Darby canine kidney cells and human colon cancer-derived Caco-2 cells. In short-hairpin RNA-based gene knockdown experiments, we show that the apical membrane targeting of CD320 is mediated by a Rab11a-dependent mechanism. These results extend our knowledge regarding the cell biology of CD320 and its role in vitamin B12 metabolism.

Hepsin: a multifunctional transmembrane serine protease in pathobiology.

Cell membrane-bound serine proteases are important in the maintenance of physiological homeostasis. Hepsin is a type II transmembrane serine protease highly expressed in the liver. Recent studies indicate that hepsin activates prohepatocyte growth factor in the liver to enhance Met signaling, thereby regulating glucose, lipid, and protein metabolism. In addition, hepsin functions in nonhepatic tissues, including the adipose tissue, kidney, and inner ear, to regulate adipocyte differentiation, urinary protein processing, and auditory function, respectively. In mouse models, hepsin deficiency lowers blood glucose, lipid, and protein levels, impairs uromodulin assembly in renal epithelial cells, and causes hearing loss. Elevated hepsin expression has also been found in many cancers. As a type II transmembrane protease, cell surface expression and zymogen activation are essential for hepsin activity. In this review, we discuss the current knowledge regarding hepsin biosynthesis, activation, and functions in pathobiology.

A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells.

Selective protein distribution on distinct plasma membranes is important for epithelial cell function. To date, how proteins are directed to specific epithelial cell surface is not fully understood. Here we report a conserved DSSDE motif in LDL-receptor (LDLR) modules of corin (a transmembrane serine protease) and CD320 (a receptor for vitamin B12 uptake), which regulates apical membrane targeting in renal epithelial cells. Altering this motif prevents specific apical corin and CD320 expression in polarized Madin-Darby canine kidney (MDCK) cells. Mechanistic studies indicate that this DSSDE motif participates in a Rab11a-dependent mechanism that specifies apical sorting. In MDCK cells, inhibition of Rab11a, but not Rab11b, expression leads to corin and CD320 expression on both apical and basolateral membranes. Together, our results reveal a novel molecular recognition mechanism that regulates LDLR module-containing proteins in their specific apical expression in polarized renal epithelial cells.

Intracellular autoactivation of TMPRSS11A, an airway epithelial transmembrane serine protease.

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.

N-glycan in the scavenger receptor cysteine-rich domain of hepsin promotes intracellular trafficking and cell surface expression.

The group A scavenger receptor cysteine-rich (SRCR) domain is a conserved module present in numerous proteins involved in diverse biological processes. Hepsin, a hepatic protease implicated in many cancers, consists of a cytoplasmic tail, a transmembrane domain and an extracellular regions with a group A SRCR domain and a serine protease domain. Like in many SRCR-containing proteins, the SRCR domain in hepsin has an N-glycosylation site, but its functional significance is unknown. In this study, we confirmed N-glycosylation at Asn112 in hepsin by glycosidase digestion and site-directed mutagenesis in human hepatoma cells. In Western blotting, fluorogenic substrate assay, flow cytometry, and protein-chase experiments, we found that Asn112 to Gln (N112Q) mutation inhibited hepsin intracellular trafficking, cell surface expression, and zymogen activation. By immunofluorescent staining, we found that the N112Q mutant was more abundant than wild-type hepsin in the endoplasmic reticulum (ER). Further co-immunoprecipitation studies indicated increased binding of the N112Q mutant to calnexin and binding-immunoglobulin protein (BiP), two ER chaperones. Our results indicate that the N-glycan in the SRCR domain of hepsin promotes intracellular trafficking and cell surface expression, possibly by a calnexin-dependent mechanism in facilitating ER exiting.

Autoactivation and calpain-1-mediated shedding of hepsin in human hepatoma cells.

Hepsin is a transmembrane serine protease implicated in many biological processes, including hepatocyte growth, urinary protein secretion, auditory nerve development, and cancer metastasis. Zymogen activation is critical for hepsin function. To date, how hepsin is activated and regulated in cells remains an enigma. In this study, we conducted site-directed mutagenesis, cell expression, plasma membrane protein labeling, trypsin digestion, Western blotting, and flow cytometry experiments in human hepatoma HepG2 cells, where hepsin was originally discovered, and SMMC-7721 cells. Our results show that hepsin is activated by autocatalysis on the cell surface but not intracellularly. Moreover, we show that hepsin undergoes ectodomain shedding. In the conditioned medium from HepG2 and SMMC-7721 cells, we detected a soluble fragment comprising nearly the entire extracellular region of hepsin. By testing protease inhibitors, gene knockdown, and site-directed mutagenesis, we identified calpain-1 as a primary protease that acted extracellularly to cleave Tyr52 in the juxtamembrane space of hepsin. These results provide new insights into the biochemical and cellular mechanisms that regulate hepsin expression and activity.

The effect of different types of abdominal binders on intra-abdominal pressure.

OBJECTIVES: To investigate the effect of non-elastic/elastic abdominal binders on intra-vesical pressure (IVP), physiological functions, and clinical outcomes in laparotomy patients at the perioperative stage.  METHODS: This prospective study was conducted from May to October 2014 at the Trauma Surgery Department, Daping Hospital, Chongqing, China. Laparotomy patients were randomly divided into non-elastic abdominal binder group (28 patients), and elastic abdominal binder group (29 patients). Binders were applied for 14 days following the operation, or until discharge. Demographic information, Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation  II (APACHE-II) scores (prior to the operation, on the first day after operation, the day IVP measurement was stopped, and one day before discharge), and outcomes were recorded. The IVP was measured before the operation to postoperative day 7. RESULTS: There were no significant differences in the demographic information, outcomes, SOFA or APACHE-II scores between the 2 groups. Initial out-of-bed mobilization occurred earlier in the elastic binder group (3.2 ± 2.0 versus 5.0 ± 3.7 days, p=0.028). A greater increase in IVP was observed in the non-elastic binder group than in the elastic binder group (2.9 ± 1.1 versus 1.1 ± 0.7 mm Hg, p=0.000). CONCLUSION: Elastic binders have relatively little effect on IVP and are more helpful at promoting postoperative recovery than non-elastic binders. Therefore, elastic binders are more suitable for clinical use.

"One-stop hybrid procedure" in the treatment of vascular injury of lower extremity.

As a new surgical technique, "one-stop hybrid procedure" is rarely applied in trauma patients. This paper aims to explore its role in vascular injury of the lower extremity. Vascular intervention combined with open surgery was performed to treat three cases of vessel injuries of the lower extremity in our hybrid operating room. One patient with stab injury to the left femoral vein was treated by temporary artery blocking after excluding arterial injury by angiography, followed by blocking surgery and debridement and repair of the injured vein. The other two patients with drug addiction history, who were found to have pricking injuries to the femoral artery combined with local infection, were successfully treated by endovascular techniques and open debridement. One-stop hybrid procedure in treating vascular injury patients could simplify the operation procedure, reduce operative risk, and achieve good curative effect.

A Ser252Trp mutation in fibroblast growth factor receptor 2 (FGFR2) mimicking human Apert syndrome reveals an essential role for FGF signaling in the regulation of endochondral bone formation.

A S252W mutation of fibroblast growth factor receptor 2 (FGFR2), which is responsible for nearly two-thirds of Apert syndrome (AS) cases, causes retarded development of the skeleton and skull malformation resulting from premature fusion of the craniofacial sutures. We utilized a Fgfr2(+/S252W) mouse (a knock-in mouse model mimicking human AS) to demonstrate decreased bone mass due to reduced trabecular bone volume, reduced bone mineral density, and shortened growth plates in the long bones. In vitro bone mesenchymal stem cells (BMSCs) culture studies revealed that the mutant mice showed reduced BMSC proliferation, a reduction in chondrogenic differentiation, and reduced mineralization. Our results suggest that these phenomena are caused by up-regulation of p38 and Erk1/2 phosphorylation. Treatment of cultured mutant bone rudiments with SB203580 or PD98059 resulted in partial rescue of the bone growth retardation. The p38 signaling pathway especially was found to be responsible for the retarded long bone development. Our data indicate that the S252W mutation in FGFR2 directly affects endochondral ossification, resulting in growth retardation of the long bone. We also show that the p38 and Erk1/2 signaling pathways partially mediate the effects of the S252W mutation of FGFR2 on long bone development.

Stromal cell-derived factor-1 receptor CXCR4-overexpressing bone marrow mesenchymal stem cells accelerate wound healing by migrating into skin injury areas.

Stromal cell-derived factor-1 (SDF-1) and its membrane receptor C-X-C chemokine receptor type 4 (CXCR4) are involved in the homing and migration of multiple stem cell types, neovascularization, and cell proliferation. This study investigated the hypothesis that bone marrow-derived mesenchymal stem cells (BMSCs) accelerate skin wound healing in the mouse model by overexpression of CXCR4 in BMSCs. We compared SDF-1 expression and skin wound healing times of BALB/c mice, severe combined immunodeficiency (SCID) mice, and immune system-deficient nude mice after (60)Co radiation-induced injury of their bone marrow. The occurrence of transplanted adenovirus-transfected CXCR4-overexpressing male BMSCs in the wound area was compared with the occurrence of untransfected male BALB/c BMSCs in (60)Co-irradiated female mice skin wound healing areas by Y chromosome marker analyses. The wound healing time of BALB/c mice was 14.00±1.41 days, whereas for the nude and SCID mice it was 17.16±1.17 days and 19.83±0.76 days, respectively. Male BMSCs could be detected in the surrounding areas of (60)Co-irradiated female BALB/c mice wounds, and CXCR4-overexpressing BMSCs accelerated the wound healing time. CXCR4-overexpressing BMSCs migrate in an enhanced manner to skin wounds in a SDF-1-expression-dependent manner, thereby reducing the skin wound healing time.

Abscess formation in vertebral canal and presacral area following penetrating injury of rectum and sacral vertebra by a steel rod.

Penetrating injury to the rectum, vertebral body and spinal cord by a steel rod is a rare condition. Treatment of this kind of injury is very challenging. Rectal injury requires repair and fecal diversion, while debridement of the spine is difficult, especially when the injury site is very long. Here we report a case of penetrating injury of rectum and sacral vertebra by a steel rod after falling onto the ground from 1 m height. The abscess cavity was irrigated with 3% hydrogen peroxide and physio-logical saline repeatedly. The bony canal was carefully debrided, curetted and bony fragments were removed. Spinal irrigation and drainage lasted for 2 months and sensitive antibiotic (amikacin sulfate) was given 7 days after surgery, but abscess was still formed in the vertebral canal. At 6-month follow-up, the patient was paralyzed without any neurological improvement, and the pain in low back and lower limb still continued.

Adipose-derived stem cells modified genetically in vivo promote reconstruction of bone defects.

AIMS: Bone defects induced by different causes are difficult to replace and repair. We sought to repair bone defects by transplantation of genetically modified adipose-derived stem cells (ADSC) and acellular bone matrix (ACBM). METHODS: We constructed the biologic material of ACBM and evaluated its mechanical properties, general biocompatibility and biosafety. ADSC isolated from minipigs were cultured in vitro and then transfected by recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF) plasmids, respectively. Subsequently, the compounds of ACBM/ADSC/rhBMP-2/rhVEGF were used to repair bone defects of the ulna in minipigs. X-ray examination, radionuclide bone imaging and single photon emission computerized tomography (SPECT) were employed to monitor the therapeutic effects 2, 4, 8 and 12 weeks after operation. Histologic experiments were carried out 12 weeks after operation. RESULTS: ACBM had no or weak antigenicity and the natural mechanical properties of ACBM were preserved. In vitro, ADSC transfected by rhBMP-2 and rhVEGF, respectively, could release rhBMP-2 or rhVEGF for at least 4 weeks. The X-ray, radionuclide bone imaging and SPECT examinations indicated that the compound of ACBM/ADSC/rhBMP-2/rhVEGF had better treatment effects on bone defects compared with the controls. CONCLUSIONS: Scaffolds, seed cells and bioactive factors are key points in tissue engineering. This research indicates that ACBM is a good biologic material for tissue repair, and ACBM/ADSC/rhBMP-2/rhVEGF can accelerate bone formation significantly.