The PRY/SPRY/B30.2 Domain of Butyrophilin 1A1 (BTN1A1) Binds to Xanthine Oxidoreductase: IMPLICATIONS FOR THE FUNCTION OF BTN1A1 IN THE MAMMARY GLAND AND OTHER TISSUES*

Butyrophilin 1A1 (BTN1A1) and xanthine oxidoreductase (XOR) are highly expressed in the lactating mammary gland and are secreted into milk associated with the milk fat globule membrane (MFGM). Ablation of the genes encoding either protein causes severe defects in the secretion of milk lipid droplets, suggesting that the two proteins may function in the same pathway. Therefore, we determined whether BTN1A1 and XOR directly interact using protein binding assays, surface plasmon resonance analysis, and gel filtration. Bovine XOR bound with high affinity in a pH- and salt-sensitive manner (K_D = 101 ± 31 nm in 10 mm HEPES, 150 mm NaCl, pH 7.4) to the PRY/SPRY/B30.2 domain in the cytoplasmic region of bovine BTN1A1. Binding was stoichiometric, with one XOR dimer binding to either two BTN1A1 monomers or one dimer. XOR bound to BTN1A1 orthologs from mice, humans, or cows but not to the cytoplasmic domains of the closely related human paralogs, BTN2A1 or BTN3A1, or to the B30.2 domain of human RoRet (TRIM 38), a protein in the TRIM family. Analysis of the protein composition of the MFGM of wild type and BTN1A1 null mice showed that most of the XOR in mice lacking BTN1A1 was released from the MFGM in a soluble form when the milk lipid droplets were disrupted to prepare membrane, compared with wild-type mice, in which most of the XOR remained membrane-bound. Thus BTN1A1 functions in vivo to stabilize the association of XOR with the MFGM by direct interactions through the PRY/SPRY/B30.2 domain. The potential significance of BTN1A1/XOR interactions in the mammary gland and other tissues is discussed.Members of the butyrophilin (BTN)³ gene family are attracting increasing attention because they may play multifunctional roles in diverse physiologies, including lactation (1, 2), selection and regulation of T-cells in the immune system (3–6), and modulation of autoimmune disease (7–9). BTN proteins have the canonical structures of cell surface receptors, which, after an N-terminal signal sequence, generally comprise two exoplasmic Ig folds (10, 11), a membrane anchor and a cytoplasmic domain consisting of a stem region, a PRY/SPRY/B30.2 domain (12, 13), and a cytoplasmic tail at the C terminus (14).The eponymous BTN1A1 protein has been linked to the secretion of milk lipid droplets because it is highly expressed in the mammary epithelium during lactation and is incorporated into the surface membrane coat surrounding cytoplasmic lipid droplets (the milk fat globule membrane (MFGM)) as they bud into milk from the apical surface (15). Furthermore, ablation of the Btn1a1 gene disrupts lipid secretion, causing the accumulation of large pools of triacylglycerol in the cytoplasm of Btn1a1 null mice (1). In a different context, dietary exposure to BTN1A1 in dairy products has been associated with modulation of the autoimmune disease multiple sclerosis because of structural similarities between the IgI fold of BTN1A1 (16) and the IgV fold of myelin oligodendrocyte glycoprotein (MOG) (17) an antigen on the myelin nerve sheath that is a target for autoantibodies in multiple sclerosis patients (8–10).Potential interactions between the exoplasmic Ig folds of several BTN proteins, and putative receptors on immune cells are postulated to regulate positive selection of epidermal γδ-T cells in the case of Skint1 (6) and suppress T-cell activation in the case of BTNL2 (4, 5). In addition, BTN2A1 binds to the C-type lectin, DC-SIGN, on immature dendritic cells (18), and proteins in the BTN3A1–3 subfamily bind to an unidentified ligand on various immune cells (19).Interactions between the cytoplasmic domain of BTN and intracellular proteins have not been investigated in any detail. The intracellular region of most BTNs is dominated by the B30.2 or the PRY/SPRY domain, which comprises two sheets of antiparallel β-strands folded into a β- sandwich, which in some proteins is contiguous at the N terminus with one or two α-helices (20–24) (for a discussion of the relationship between PRY, SPRY, and B30.2 domains, see Ref. (25)). This domain (here abbreviated as B30.2),⁴ is postulated to serve as a protein-binding module, by which proteins interact through the extended surface loops that adjoin individual β-strands (22).One protein that may bind to the cytoplasmic region of BTN proteins (and the B30.2 domain) is the redox enzyme, xanthine oxidoreductase (XOR),⁵ because it was shown to bind to the cytoplasmic domain of mouse BTN1A1 in an in vitro binding assay (26). Furthermore, one XOR-deficient mouse strain (Xdh^+/−) (27) displayed a lactation phenotype similar to that of Btn1a1 null mice (1), suggesting that the two proteins may be functionally linked by direct interaction. These conclusions, however, have been challenged, because XOR does not co-localize with BTN1A1 in immunolabeled freeze-fractured replicates of secreted milk lipid droplets (28), and a second mouse strain deficient in XOR does not appear to have an altered lactation phenotype (29).In this paper, we devise in vivo and in vitro assays to show that the cytoplasmic domain of BTN1A1 binds to XOR via the B30.2 domain and that BTN1A1 is required for the stable association of XOR with the MFGM in vivo. Furthermore, interaction with XOR appears to be limited to BTN1A1 orthologs. These results are discussed in the context of potential functions of BTN1A1 in the mammary gland and other tissues and the relationship of BTN1A1 to other BTN family members.     

Plasmid replication initiator RepB forms a hexamer reminiscent of ring helicases and has mobile nuclease domains

RepB initiates plasmid rolling‐circle replication by binding to a triple 11‐bp direct repeat (bind locus) and cleaving the DNA at a specific distant site located in a hairpin loop within the nic locus of the origin. The structure of native full‐length RepB reveals a hexameric ring molecule, where each protomer has two domains. The origin‐binding and catalytic domains show a three‐layer α–β–α sandwich fold. The active site is positioned at one of the faces of the β‐sheet and coordinates a Mn²⁺ ion at short distance from the essential nucleophilic Y99. The oligomerization domains (ODs), each consisting of four α‐helices, together define a compact ring with a central channel, a feature found in ring helicases. The toroidal arrangement of RepB suggests that, similar to ring helicases, it encircles one of the DNA strands during replication to confer processivity to the replisome complex. The catalytic domains appear to be highly mobile with respect to ODs. This mobility may account for the adaptation of the protein to two distinct DNA recognition sites.     

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage

Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells. They hold an enormous therapeutic potential, but at the moment there is little information on the properties of MSCs, including their surface structures. In the present study, we analyzed the mesenchymal stem cell glycome by using mass spectrometric profiling as well as a panel of glycan binding proteins. Structural verifications were obtained by nuclear magnetic resonance spectroscopy, mass spectrometric fragmentation, and glycosidase digestions. The MSC glycome was compared to the glycome of corresponding osteogenically differentiated cells. More than one hundred glycan signals were detected in mesenchymal stem cells and osteoblasts differentiated from them. The glycan profiles of MSCs and osteoblasts were consistently different in biological replicates, indicating that stem cells and osteoblasts have characteristic glycosylation features. Glycosylation features associated with MSCs rather than differentiated cells included high-mannose type N-glycans, linear poly-N-acetyllactosamine chains and α2-3-sialylation. Mesenchymal stem cells expressed SSEA-4 and sialyl Lewis x epitopes. Characteristic glycosylation features that appeared in differentiated osteoblasts included abundant sulfate ester modifications. The results show that glycosylation analysis can be used to evaluate MSC differentiation state.     

A cross-phyla response to Daphnia chemical alarm substances by an aquatic oligochaete

Although chemical alarm substances from damaged heterospecifics have been well documented to induce defense mechanisms in potential prey, data about antipredator responses to alarm cues from prey organisms of a distinct phylum are scarce. In this study, we analyze the response of an oligochaete to chemical alarm substances from distantly related cladocerans. We conducted laboratory experiments to investigate whether the aquatic oligochaete Stylaria lacustris detects and responds to alarm substances from Daphnia. The oligochaetes were exposed to alarm substances from Daphnia magna, which is a member of the same prey guild, and D. hyalina, which is vulnerable to different predators. S. lacustris increased the fission rate in response to cues from conspecifics and the heterospecific D. magna. These species share common predators, especially damselfly larvae and hydras. In contrast, chemical cues released from D. hyalina did not induce an increase in the reproductive rate of the oligochaetes. D. hyalina is a pelagic species and is not included in the diet of predators preying upon S. lacustris. Our results indicate cross-phyla responses among invertebrates (Annelida and Arthropoda), and suggest strong selection pressures to respond to heterospecific alarm substances. The oligochaete S. lacustris is able to differentiate chemical substances released from damaged Daphnia of two different species. These results show that alarm cues released from damaged Daphnia may be species-specific. However, further studies on the chemical structure of alarm substances in cladocerans are needed to support this hypothesis.     

The Group III Two-Component Histidine Kinase of Filamentous Fungi Is Involved in the Fungicidal Activity of the Bacterial Polyketide Ambruticin

We have shown that the plant pathogen Alternaria brassicicola exhibited very high susceptibility to ambruticin VS4 and to a lesser extent to the phenylpyrrole fungicide fludioxonil. These compounds are both derived from natural bacterial metabolites with antifungal properties and are thought to exert their toxicity by interfering with osmoregulation in filamentous fungi. Disruption of the osmosensor group III histidine kinase gene AbNIK1 (for A. brassicola NIK1) resulted in high levels of resistance to ambruticin and fludioxonil, while a mutant isolate characterized by a single-amino-acid substitution in the HAMP domain of the kinase only exhibited moderate resistance. Moreover, the natural resistance of Saccharomyces cerevisiae to these antifungal molecules switched to sensitivity in strains expressing AbNIK1p. We also showed that exposure to fludioxonil and ambruticin resulted in abnormal phosphorylation of a Hog1-like mitogen-activated protein kinase (MAPK) in A. brassicicola. Parallel experiments carried out with wild-type and mutant isolates of Neurospora crassa revealed that, in this species, ambruticin susceptibility was dependent on the OS1-RRG1 branch of the phosphorelay pathway downstream of the OS2 MAPK cascade but independent of the yeast Skn7-like response regulator RRG2. These results show that the ability to synthesize a functional group III histidine kinase is a prerequisite for the expression of ambruticin and phenylpyrrole susceptibility in A. brassicicola and N. crassa and that, at least in the latter species, improper activation of the high-osmolarity glycerol-related pathway could explain their fungicidal properties.     

A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis

Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[³²P]pyrophosphate (PP_i) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (P_i) concentrations after degradation by inorganic pyrophosphatase of the PP_i released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A₄, one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[³²P]PP_i exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover.     

Golgi targeting of Drosophila melanogaster β4GalNAcTB requires a DHHC protein family–related protein as a pilot

Drosophila melanogaster β4GalNAcTB mutant flies revealed that this particular N-acetylgalactosaminyltransferase is predominant in the formation of lacdiNAc (GalNAcβ1,4GlcNAc)-modified glycolipids, but enzymatic activity could not be confirmed for the cloned enzyme. Using a heterologous expression cloning approach, we isolated β4GalNAcTB together with β4GalNAcTB pilot (GABPI), a multimembrane-spanning protein related to Asp-His-His-Cys (DHHC) proteins but lacking the DHHC consensus sequence. In the absence of GABPI, inactive β4GalNAcTB is trapped in the endoplasmic reticulum (ER). Coexpression of β4GalNAcTB and GABPI generates the active enzyme that is localized together with GABPI in the Golgi. GABPI associates with β4GalNAcTB and, when expressed with an ER retention signal, holds active β4GalNAcTB in the ER. Importantly, treatment of isolated membrane vesicles with Triton X-100 disturbs β4GalNAcTB activity. This phenomenon occurs with multimembrane-spanning glycosyltransferases but is normally not a property of glycosyltransferases with one membrane anchor. In summary, our data provide evidence that GABPI is required for ER export and activity of β4GalNAcTB.