Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Washington University School of Medicine

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

La famille chlorure de canaux activ&eacute;s par le calcium (CLCA) est des prot&eacute;ines s&eacute;cr&eacute;t&eacute;es qui r&eacute;gulent les deux expression chlorure de transport et de la mucine, contr&ocirc;lant ainsi la production de mucus dans les syst&egrave;mes respiratoires et autres. En cons&eacute;quence, la CLCA1 humaine est un m&eacute;diateur critique de hypers&eacute;cr&eacute;toire maladies pulmonaires, telles que l&#39;asthme, maladie pulmonaire obstructive chronique et la fibrose kystique, qui manifeste l&#39;obstruction de la glaire. Malgr&eacute; la pertinence de l&#39;hom&eacute;ostasie et de la maladie, le m&eacute;canisme de CLCA1 fonction reste en grande partie non d&eacute;fini. Nous combler ce vide en montrant que les prot&eacute;ines CLCA contiennent un site de clivage prot&eacute;olytique de consensus reconnu par un roman zincin m&eacute;talloprot&eacute;ase domaine situ&eacute; &agrave; l&#39;extr&eacute;mit&eacute; N terminale de CLCA lui-m&ecirc;me. CLCA1 des mutations qui inhibent self-clivage emp&ecirc;chent l&#39;activation des canaux activ&eacute;s par le calcium chlorure (CaCC)-transport de chlorure de m&eacute;diation. CaCC activation n&eacute;cessite un clivage pour d&eacute;masquer le fragment N-terminal de CLCA1, qui peut ind&eacute;pendamment porte CaCCs. Blocage de CaCCs m&eacute;di&eacute;e par CLCA1 ne semble pas impliquer le clivage prot&eacute;olytique de la cha&icirc;ne parce qu&#39;un mutant fragment N-terminal d&eacute;ficient en activit&eacute; prot&eacute;olytique est capable d&#39;induire des courants comparables &agrave; celle du fragment natif. Ces donn&eacute;es fournissent une mani&egrave;re m&eacute;caniste pour CLCA1 soi-clivage et un nouveau m&eacute;canisme de r&eacute;gulation de l&#39;activit&eacute; de canal chlorure sp&eacute;cifique &agrave; l&#39;interface de muqueuse.

Self-clivage de la prot&eacute;ine humaine de CLCA1 par un domaine roman m&eacute;talloprot&eacute;ase interne contr&ocirc;le l&#39;activation de canaux activ&eacute;s par le calcium chlorure.

La famiglia di calcio-attivato (CLCA) canale di cloruro &egrave; proteine secrete che regolano entrambi espressione cloruro di trasporto e mucina, controllando cos&igrave; la produzione di muco in sistemi respiratorio e altri. Di conseguenza, CLCA1 umana &egrave; un mediatore critico si malattie polmonari, come l&#39;asma, malattia polmonare ostruttiva cronica e fibrosi cistica, che manifestano ostruzione di muco. Nonostante la rilevanza per l&#39;omeostasi e malattia, rimane in gran parte definito il meccanismo della funzione CLCA1. Affrontiamo questo vuoto mostrando che le proteine CLCA contengono un sito di clivaggio proteolitico consenso riconosciuto da un romanzo zincin metalloprotease dominio che si trova all&#39;interno di capolinea N di CLCA stesso. CLCA1 mutazioni che inibiscono self-scissione evitare attivazione del canale del cloro attivati dal calcio (CaCC)-mediata trasporto di cloruro. CaCC attivazione richiede clivaggio a smascherare il frammento N-terminale del CLCA1, che pu&ograve; autonomamente gate CaCCs. Sbarramento di CaCCs mediata da CLCA1 non sembra coinvolgere il clivaggio proteolitico del canale perch&eacute; un frammento N-terminale carente di attivit&agrave; proteolitica mutante &egrave; in grado di indurre correnti comparabile con quella del frammento nativo. Questi dati forniscono una base meccanicistica per CLCA1 auto-fenditura sia un nuovo meccanismo per la regolazione dell&#39;attivit&agrave; del canale del cloruro specifiche per l&#39;interfaccia della mucosa.

Self-scissione delle proteine umane CLCA1 da un dominio romanzo metalloprotease interno controlla attivazione canale cloruro di calcio-attivato.

Calcium-activated chloride channel regulator 1 (CLCA1) activates calcium-dependent chloride currents; neither the target, nor mechanism, is known. We demonstrate that secreted CLCA1 activates calcium-dependent chloride currents in HEK293T cells in a paracrine fashion, and endogenous TMEM16A/Anoctamin1 conducts the currents. Exposure to exogenous CLCA1 increases cell surface levels of TMEM16A and cellular binding experiments indicate CLCA1 engages TMEM16A on the surface of these cells. Altogether, our data suggest that CLCA1 stabilizes TMEM16A on the cell surface, thus increasing surface expression, which results in increased calcium-dependent chloride currents. Our results identify the first Cl(-) channel target of the CLCA family of proteins and establish CLCA1 as the first secreted direct modifier of TMEM16A activity, delineating a unique mechanism to increase currents. These results suggest cooperative roles for CLCA and TMEM16 proteins in influencing the physiology of multiple tissues, and the pathology of multiple diseases, including asthma, COPD, cystic fibrosis, and certain cancers.

Secreted CLCA1 modulates TMEM16A to activate Ca(2+)-dependent chloride currents in human cells.

The p38 MAP kinases (p38 MAPKs) represent an important family centrally involved in mediating extracellular signaling. Recent studies indicate that family members such as MAPK13 (p38δ) display a selective cellular and tissue expression and are therefore involved in specific diseases. Detailed structural studies of all p38 MAPK family members are crucial for the design of specific inhibitors. In order to facilitate such ventures, the structure of MAPK13 was determined in both the inactive (unphosphorylated; MAPK13) and active (dual phosphorylated; MAPK13/pTpY) forms. Here, the first preparation, crystallization and structure determination of MAPK13/pTpY are presented and the structure is compared with the previously reported structure of MAPK13 in order to facilitate studies for structure-based drug design. A comprehensive analysis of inactive versus active structures for the p38 MAPK family is also presented. It is found that MAPK13 undergoes a larger interlobe configurational rearrangement upon activation compared with MAPK14. Surprisingly, the analysis of activated p38 MAPK structures (MAP12/pTpY, MAPK13/pTpY and MAPK14/pTpY) reveals that, despite a high degree of sequence similarity, different side chains are used to coordinate the phosphorylated residues. There are also differences in the rearrangement of the hinge region that occur in MAPK14 compared with MAPK13 which would affect inhibitor binding. A thorough examination of all of the active (phosphorylated) and inactive (unphosphorylated) p38 MAPK family member structures was performed to reveal a common structural basis of activation for the p38 MAP kinase family and to identify structural differences that may be exploited for developing family member-specific inhibitors.

The crystal structure of phosphorylated MAPK13 reveals common structural features and differences in p38 MAPK family activation.

Chloride transport proteins play critical roles in inflammatory airway diseases, contributing to the detrimental aspects of mucus overproduction, mucus secretion, and airway constriction. However, they also play crucial roles in contributing to the innate immune properties of mucus and mucociliary clearance. In this review, we focus on the emerging novel roles for a chloride channel regulator (CLCA1), a calcium-activated chloride channel (TMEM16A), and two chloride exchangers (SLC26A4/pendrin and SLC26A9) in chronic inflammatory airway diseases.

Zeynep Yurtsever

Department of Internal Medicine,
Drug Discovery Program in Pulmonary and Critical Care Medicine,
Biochemistry Program,
Center for the Investigation of Membrane Excitability Diseases

Washington University School of Medicine

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Zeynep Yurtsever

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Internal Medicine,Drug Discovery Program in Pulmonary and Critical Care Medicine,Biochemistry Program,Center for the Investigation of Membrane Excitability Diseases

Washington University School of Medicine

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Department of Internal Medicine,
Drug Discovery Program in Pulmonary and Critical Care Medicine,
Biochemistry Program,
Center for the Investigation of Membrane Excitability Diseases