scholarly journals Biochemical and Molecular Characterization of a Novel Type of Mutanase from Paenibacillus sp. Strain RM1: Identification of Its Mutan-Binding Domain, Essential for Degradation of Streptococcus mutans Biofilms

2008 ◽  
Vol 74 (9) ◽  
pp. 2759-2765 ◽  
Author(s):  
Isao Shimotsuura ◽  
Hiromitsu Kigawa ◽  
Motoyasu Ohdera ◽  
Howard K. Kuramitsu ◽  
Syozi Nakashima
Keyword(s):  
Streptococcus Mutans ◽  
Structural Integrity ◽  
Binding Activity ◽  
Etiologic Agent ◽  
Binding Domain ◽  
Intact Protein ◽  
Calculated Molecular Mass ◽  
Terminal Domain ◽  
Paenibacillus Sp

ABSTRACT A novel type of mutanase (termed mutanase RM1) was isolated from Paenibacillus sp. strain RM1. The purified enzyme specifically hydrolyzed α-1,3-glucan (mutan) and effectively degraded biofilms formed by Streptococcus mutans, a major etiologic agent in the progression of dental caries, even following brief incubation. The nucleotide sequence of the gene for this protein contains a 3,873-bp open reading frame encoding 1,291 amino acids with a calculated molecular mass of 135 kDa. The protein contains two major domains, the N-terminal domain (277 residues) and the C-terminal domain (937 residues), separated by a characteristic sequence composed of proline and threonine repeats. The characterization of the recombinant proteins for each domain which were expressed in Escherichia coli demonstrated that the N-terminal domain had strong mutan-binding activity but no mutanase activity whereas the C-terminal domain was responsible for mutanase activity but had mutan-binding activity significantly lower than that of the intact protein. Importantly, the biofilm-degrading activity observed with the intact protein was not exhibited by either domain alone or in combination with the other. Therefore, these results indicate that the structural integrity of mutanase RM1 containing the N-terminal mutan-binding domain is required for the biofilm-degrading activity.

scholarly journals Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA ReplicationIn Vivo

2016 ◽  
Vol 90 (6) ◽  
pp. 3198-3211 ◽  
Author(s):  
Monika Bergvall ◽  
David Gagnon ◽  
Steve Titolo ◽  
Michaël Lehoux ◽  
Claudia M. D'Abramo ◽  
...  
Keyword(s):  
Dna Replication ◽  
Structural Integrity ◽  
Alpha Helix ◽  
Helicase Domain ◽  
Atp Binding Site ◽  
Terminal Domain ◽  
Replication Activity

ABSTRACTThe papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose functionin vivois still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1in vitroand confirmed herein vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1in vivoand suggest that the function of the C-tail has evolved in a PV type-specific manner.IMPORTANCEWhile much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1in vivoand that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural integrity of the helicase domain, followed by an acidic region (AR) and a C-terminal tail (C-tail) that have been shown to regulate the oligomerization of BPV1 E1in vitro. Characterization of E1 chimeras revealed that, while the function of the AR could be transferred from BPV1 E1 to HPV11 E1, that of the C-tail could not. These results suggest that the E1 CTD performs multiple functions in DNA replication, some of them in a virus type-specific manner.

Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins

2001 ◽  
Vol 114 (1) ◽  
pp. 161-172 ◽  
Author(s):  
D. Sun ◽  
C.L. Leung ◽  
R.K. Liem
Keyword(s):  
Calcium Binding ◽  
Binding Domain ◽  
Actin Binding ◽  
Microtubule Binding ◽  
Transfected Cells ◽  
Terminal Domain ◽  
Microtubule Binding Domain

MACF (microtubule actin cross-linking factor) is a large, 608-kDa protein that can associate with both actin microfilaments and microtubules (MTs). Structurally, MACF can be divided into 3 domains: an N-terminal domain that contains both a calponin type actin-binding domain and a plakin domain; a rod domain that is composed of 23 dystrophin-like spectrin repeats; and a C-terminal domain that includes two EF-hand calcium-binding motifs, as well as a region that is homologous to two related proteins, GAR22 and Gas2. We have previously demonstrated that the C-terminal domain of MACF binds to MTs, although no homology was observed between this domain and other known microtubule-binding proteins. In this report, we describe the characterization of this microtubule-binding domain of MACF by transient transfection studies and in vitro binding assays. We found that the C-terminus of MACF contains at least two microtubule-binding regions, a GAR domain and a domain containing glycine-serine-arginine (GSR) repeats. In transfected cells, the GAR domain bound to and partially stabilized MTs to depolymerization by nocodazole. The GSR-containing domain caused MTs to form bundles that are still sensitive to nocodazole-induced depolymerization. When present together, these two domains acted in concert to bundle MTs and render them stable to nocodazole treatment. Recently, a study has shown that the N-terminal half of the plakin domain (called the M1 domain) of MACF also binds MTs. We therefore examined the microtubule binding ability of the M1 domain in the context of the entire plakin domain with and without the remaining N-terminal regions of two different MACF isoforms. Interestingly, in the presence of the surrounding sequences, the M1 domain did not bind MTs. In addition to MACF, cDNA sequences encoding the GAR and GSR-containing domains are also found in the partial human EST clone KIAA0728, which has high sequence homology to the 3′ end of the MACF cDNA; hence, we refer to it as MACF2. The C-terminal domain of mouse MACF2 was cloned and characterized. The microtubule-binding properties of MACF2 C-terminal domain are similar to that of MACF. The GAR domain was originally found in Gas 2 protein and here we show that it can associate with MTs in transfected cells. Plectin and desmoplakin have GSR-containing domains at their C-termini and we further demonstrate that the GSR-containing domain of plectin, but not desmoplakin, can bind to MTs in vivo.

scholarly journals Porcine Arterivirus Attachment to the Macrophage-Specific Receptor Sialoadhesin Is Dependent on the Sialic Acid-Binding Activity of the N-Terminal Immunoglobulin Domain of Sialoadhesin

2007 ◽  
Vol 81 (17) ◽  
pp. 9546-9550 ◽  
Author(s):  
Peter L. Delputte ◽  
Wander Van Breedam ◽  
Iris Delrue ◽  
Cornelia Oetke ◽  
Paul R. Crocker ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Binding Activity ◽  
Sialic Acids ◽  
Binding Domain ◽  
Respiratory Syndrome Virus ◽  
Specific Receptor ◽  
Terminal Domain ◽  
Sialic Acid Binding ◽  
Immunoglobulin Domain ◽  
Binding Lectin

ABSTRACT The sialic acid-binding lectin sialoadhesin (Sn) is a macrophage-restricted receptor for porcine reproductive and respiratory syndrome virus (PRRSV). To investigate the importance of pSn sialic acid-binding activity for PRRSV infection, an R116-to-E mutation was introduced in the predicted sialic acid-binding domain of pSn, resulting in a mutant, pSnRE, that could not bind sialic acids. PSn, but not pSnRE, allowed PRRSV binding and internalization. These data show that the sialic acid-binding activity of pSn is essential for PRRSV attachment to pSn and thus identifies the variable, N-terminal domain of Sn as a PRRSV binding domain.

scholarly journals Purification and characterization of a saliva-interacting cell-wall protein from Streptococcus mutans serotype f by using monoclonal-antibody immunoaffinity chromatography

1985 ◽  
Vol 228 (1) ◽  
pp. 211-217 ◽  
Author(s):  
F Ackermans ◽  
J P Klein ◽  
J Ogier ◽  
H Bazin ◽  
F Cormont ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Streptococcus Mutans ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoaffinity Chromatography ◽  
Binding Activity ◽  
Immunoglobulin M ◽  
Single Polypeptide Chain

A rat monoclonal antibody, LO SM2, of the immunoglobulin M class, specific for a saliva receptor (SR) from Streptococcus mutans serotype f, was able to precipitate the SR from crude cell-wall-associated antigens (WEA) of this bacteria in presence of a detergent mixture. We have then used the technique of monoclonal-antibody immunoaffinity chromatography to purify the S. mutans SR. Pure SR was obtained from a crude WEA fraction with a single chromatographic step. The active SR could be eluted from the column in a highly purified form with 0.2 M-glycine/HC1, pH 2.8. The final yield was about 32% in terms of binding activity. Characterization of the SR by crossed immunoelectrophoresis, sodium dodecyl sulphate- or 4-30%-native-gradient-polyacrylamide-gel electrophoresis showed that the receptor is a single polypeptide chain of Mr approx. 74000. Native or denaturated forms of the SR adsorbed on to a solid support, such as nitrocellulose, are recognized by monoclonal antibody LO SM2, and both forms are still able to bind the ligand, saliva.

scholarly journals Isolation and characterization of the flavin-binding domain of flavocytochrome b2 expressed independently in Escherichia coli

1995 ◽  
Vol 309 (2) ◽  
pp. 601-605 ◽  
Author(s):  
A Balme ◽  
C E Brunt ◽  
R L Pallister ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Escherichia Coli ◽  
Binding Domain ◽  
Flavin Mononucleotide ◽  
Exponential Process ◽  
Flavocytochrome B2 ◽  
Terminal Domain ◽  
Isolation And Characterization ◽  
Flavin Binding ◽  
Single Exponential

Flavocytochrome b2 consists of two distinct domains. The N-terminal domain contains protohaem IX and the larger, C-terminal domain contains flavin mononucleotide (FMN). We describe here the isolation of the flavin-binding domain expressed in Escherichia coli independent of the cytochrome domain. The isolated domain is an efficient lactate dehydrogenase with ferricyanide as electron acceptor but reduces cytochrome c, the physiological oxidant for flavocytochrome b2, extremely poorly; electron transfer from the flavin-binding domain to the separately expressed cytochrome domain is undetectable. FMN reduction by lactate occurs as a single exponential process in the isolated flavin-binding domain, in contrast to the biphasic kinetics observed with native flavocytochrome b2.

scholarly journals Aurora A Regulates the Activity of HURP by Controlling the Accessibility of Its Microtubule-binding Domain

2008 ◽  
Vol 19 (5) ◽  
pp. 2083-2091 ◽  
Author(s):  
Jim Wong ◽  
Robert Lerrigo ◽  
Chang-Young Jang ◽  
Guowei Fang
Keyword(s):  
Ectopic Expression ◽  
Binding Activity ◽  
Binding Domain ◽  
Aurora A ◽  
Microtubule Binding ◽  
Physiological Importance ◽  
Mitotic Kinase ◽  
Terminal Domain ◽  
Microtubule Binding Domain ◽  
And Function

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.

scholarly journals A Biochemical Characterization of the DNA Binding Activity of the Response Regulator VicR from Streptococcus mutans

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e108027 ◽  
Author(s):  
Eduardo Ayala ◽  
Jennifer S. Downey ◽  
Lauren Mashburn-Warren ◽  
Dilani B. Senadheera ◽  
Dennis G. Cvitkovitch ◽  
...  
Keyword(s):  
Dna Binding ◽  
Streptococcus Mutans ◽  
Biochemical Characterization ◽  
Response Regulator ◽  
Binding Activity ◽  
Dna Binding Activity

scholarly journals Characterization of the dextran-binding domain in the glucan-binding protein C of Streptococcus mutans

2015 ◽  
Vol 119 (4) ◽  
pp. 1148-1157 ◽  
Author(s):  
Y. Takashima ◽  
K. Fujita ◽  
A.C. Ardin ◽  
K. Nagayama ◽  
R. Nomura ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Protein C ◽  
Binding Protein ◽  
Binding Domain

Application of solid-phase immune electron microscopy to study physical and stability characteristics of enterically transmitted non-a, non-b hepatitis virus

1988 ◽  
Vol 46 ◽  
pp. 80-81
Author(s):  
Charles D. Humphrey ◽  
E. H. Cook ◽  
Karen A. McCaustland ◽  
Daniel W. Bradley
Keyword(s):  
Electron Microscopy ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Solid Phase ◽  
Etiologic Agent ◽  
World Health ◽  
Health Concern ◽  
Morphological Identification ◽  
Immune Electron Microscopy

Enterically transmitted non-A, non-B hepatitis (ET-NANBH) is a type of hepatitis which is increasingly becoming a significant world health concern. As with hepatitis A virus (HAV), spread is by the fecal-oral mode of transmission. Until recently, the etiologic agent had not been isolated and identified. We have succeeded in the isolation and preliminary characterization of this virus and demonstrating that this agent can cause hepatic disease and seroconversion in experimental primates. Our characterization of this virus was facilitated by immune (IEM) and solid phase immune electron microscopic (SPIEM) methodologies.Many immune electron microscopy methodologies have been used for morphological identification and characterization of viruses. We have previously reported a highly effective solid phase immune electron microscopy procedure which facilitated identification of hepatitis A virus (HAV) in crude cell culture extracts. More recently we have reported utilization of the method for identification of an etiologic agent responsible for (ET-NANBH).

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