Plasma membrane localization of the Toll protein in the syncytial Drosophila embryo: importance of transmembrane signaling for dorsal-ventral pattern formation

Formation of the Drosophila embryo's dorsal-ventral pattern requires the maternal product of the Toll gene. DNA sequence and genetic analyses together suggested that the Toll gene product is a transmembrane protein which communicates information from an extracytoplasmic compartment to the cytoplasm. Using antibodies as probes, we show that the Toll protein is a 135 x 10(3) Mr glycoprotein which is tightly associated with embryonic membranes. During the syncytial stage when dorsal-ventral polarity is established, the maternal Toll protein is associated with the plasma membrane around the entire embryo. During later embryonic stages, the Toll protein is expressed zygotically on many cell surfaces, possibly to promote cell adhesion. The plasma membrane localization of the Toll protein in the syncytial embryo suggests that transmembrane signaling from the extracellular perivitelline space to the cytoplasm is required for establishment of the embryonic dorsal-ventral pattern.     

Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon

Invertebrates rely completely for their protection against pathogens on the innate immune system. This non-self-recognition is activated by microbial cell wall components with unique conserved molecular patterns. Pathogen-associated molecular patterns (PAMPs) are recognised by pattern recognition receptors (PRRs). Toll and its mammalian homologs Toll-like receptors are cell-surface receptors acting as PRRs and involved in the signalling pathway implicated in their immune response. Here we describe a novel partial Toll receptor gene cloned from a gill library of the giant tiger shrimp, Penaeus monodon, using primers based on the highly conserved Toll/IL-1R (TIR) domain. The deduced amino acid sequence of the P. monodon Toll (PmToll) shows 59% similarity to a Toll-related protein of Apis mellifera. Analysis of the LRRs of shrimp Toll contained no obvious PAMP-binding insertions. Phylogenetic analysis with the insect Toll family shows clustering with Toll1 and Toll5 gene products, and it is less related to Toll3 and Toll4. Furthermore, RT-qPCR shows that PmToll is constitutively expressed in gut, gill and hepatopancreas. Challenge with white spot syndrome virus (WSSV) shows equal levels of expression in these organs. A role in the defence mechanism is discussed. In conclusion, shrimp possess at least one Toll receptor that might be involved in immune defence.     

The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase.

As a preliminary step in the understanding of the function of the Escherichia coli HtrA (DegP) protein, which is indispensable for bacterial survival only at elevated temperatures, the protein was purified and partially characterized. The HtrA protein was purified from cells carrying the htrA gene cloned into a multicopy plasmid, resulting in its overproduction. The sequence of the 13 N-terminal amino acids of the purified HtrA protein was determined and was identical to the one predicted for the mature HtrA protein by the DNA sequence of the cloned gene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrA protein is derived by cleavage of the first 26 amino acids of the pre-HtrA precursor polypeptide and that the point of cleavage follows a typical target sequence recognized by the leader peptidase enzyme. The HtrA protein was shown to be a specific endopeptidase which was inhibited by diisopropylfluorophosphate, suggesting that HtrA is a serine protease.     

Substrate specificity and ionic regulation of GlnPQ from Lactococcus lactis. An ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains

We report on the functional characterization of GlnPQ, an ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains. The first predicted transmembrane helix of GlnP was cleaved off in the mature protein and most likely serves as the signal sequence for the extracytoplasmic substrate-binding domains. Deletion analysis showed that the substrate-binding domain, in the primary sequence of GlnP nearest to the translocator domain, is used as the receptor that delivers the substrate to the translocator. Membrane reconstitution of the detergent-solubilized and purified GlnPQ complex yielded proteoliposomes that transported glutamine and glutamic acid at the expense of ATP. The transport activity of GlnPQ increased with lumenal salt concentration and internal pH, but the mechanism of ionic activation of the transporter is distinct from that of other osmoregulatory ATP-binding cassette transporters and does not depend on the presence of anionic lipids. The regulation of GlnPQ conforms to an electrostatic switch in which protein domain(s) and low molecular weight electrolytes participate.     

46 kd mannose 6-phosphate receptor: cloning, expression, and homology to the 215 kd mannose 6-phosphate receptor

We have isolated cDNA clones encoding the entire sequence of the bovine 46 kd cation-dependent mannose 6-phosphate (CD Man-6-P) receptor. Translation of CD Man-6-P receptor mRNA in Xenopus laevis oocytes results in a protein that binds specifically to phosphomannan-Sepharose, thus demonstrating that our cDNA clones encode a functional receptor. The deduced 279 amino acid sequence reveals a single polypeptide chain that contains a putative signal sequence and a transmembrane domain. Trypsin digestion of microsomal membranes containing the receptor and the location of the five potential N-linked glycosylation sites indicate that the receptor is a transmembrane protein with an extracytoplasmic amino terminus. This extracytoplasmic domain is homologous to the approximately 145 amino acid long repeating domains present in the 215 kd cation-independent Man-6-P receptor.     

丝状支原体丝状亚种SC型脂蛋白Q N末端基因的表达、纯化与抗原性分析

为了表达丝状支原体丝状亚种SC型(MmmSC)中国分离株HVRIⅩ脂蛋白Q(LppQ)N末端基因,将该基因经PCR扩增后克隆至原核表达载体pET32a中,经酶切、PCR、测序证实获得了重组表达质粒,转化Escherichia coliBL21(DE3)菌,经IPTG诱导后获得可溶性融合蛋白,表达量占菌体总蛋白的53.7%,用Ni-NTAHis.Bind纯化试剂盒纯化后,蛋白纯度达95%以上。表达蛋白经Western blot检测其抗原活性,结果表明纯化蛋白可与CBPP标准阳性血清发生强烈的反应,而与阴性血清不发生反应。     

Identification of a Linked Set of Genes in Serpulina hyodysenteriae (B204) Predicted To Encode Closely Related 39-Kilodalton Extracytoplasmic Proteins

A tandem pair of nearly identical genes from Serpulina hyodysenteriae (B204) were cloned and sequenced. The full open reading frame of one gene and the partial open reading frame of the neighboring gene appear to encode secreted proteins which are homologous to, yet distinct from, the 39-kDa extracytoplasmic protein purified from the membrane fraction of S. hyodysenteriae. We have designated these newly identified genes vspA and vspB (for variable surface protein).