Folding of carboxyl domain and assembly of procollagen I

An early form of procollagen I was found in acetic acid extracts of radioactively labeled chick embryo skull bones. It resembled native procollagen I, but sedimented slightly faster, and its component chains were slightly underhydroxylated and were not disulfide-linked to each other, although its propeptides were internally disulfide-bonded. Pulse-chase experiments showed its conversion to disulfide-linked procollagen. As the same conversion occurred when proline hydroxylation was blocked by 2,2'-dipyridyl, we infer that the formation of this precursor from its component chains does not require collagen triple helix formation. We suggest that interaction between the folded carboxyl propeptides of individual pro-alpha (I) chains is an important step in the formation of this precursor and of procollagen I. Studies of the refolding and association of fully reduced and denatured carboxyl propeptides supported this concept. In the presence of glutathione the correct disulfide bonds could be reestablished, as judged by a mapping of some tryptic peptides. Individual carboxyl propeptides refolded first, and this occurred even in 2 M urea. Recognition between folded carboxyl propeptides occurred only when less than 0.5 M urea was present. The presence of the carboxyl telopeptides was important for trimeric reassembly. Individual propeptides also folded spontaneously during cell-free translation of pro-alpha (I) chains and were recognized by specific antibodies. We consider the role of carboxyl propeptides in the formation of procollagen I molecules and suggest a model of self-assembly, possibly facilitated by interactions with the luminal surface of the rough endoplasmic reticulum.     

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.     

Motion processing streams in Drosophila are behaviorally specialized

Motion vision is an ancient faculty, critical to many animals in a range of ethological contexts, the underlying algorithms of which provide central insights into neural computation. However, how motion cues guide behavior is poorly understood, as the neural circuits that implement these computations are largely unknown in any organism. We develop a systematic, forward genetic approach using high-throughput, quantitative behavioral analyses to identify the neural substrates of motion vision in Drosophila in an unbiased fashion. We then delimit the behavioral contributions of both known and novel circuit elements. Contrary to expectation from previous studies, we find that orienting responses to motion are shaped by at least two neural pathways. These pathways are sensitive to different visual features, diverge immediately postsynaptic to photoreceptors, and are coupled to distinct behavioral outputs. Thus, behavioral responses to complex stimuli can rely on surprising neural specialization from even the earliest sensory processing stages.     

Basement membrane matrices in mouse embryogenesis, teratocarcinoma differentiation and in neuromuscular maturation

In this paper we discuss studies on basement membrane and interstitial matrix molecules in early development and teratocarcinoma differentiation. In the early embryo a compartmentalization of newly formed cell types takes place immediately by formation of basement membranes. The stage-specific developmental appearance of extracellular matrix molecules such as type IV collagen, laminin, entactin, fibronectin and proteoglycans seems to reflect a diversified role of extracellular matrices already in the earliest stages of development. In teratocarcinoma cultures the appearance and composition of extracellular matrices during the differentiation of endoderm cells closely resembles that found in the early embryo. Also in this respect the teratocarcinoma system can be used as a model for studies on early development. In later developmental phenomena other matrix molecules can also be of importance. Merosin, a novel tissue-specific basement membrane-associated protein that appears during muscle and nerve maturation is an example of such molecules.     

Drosophila basement membrane procollagen alpha 1(IV). II. Complete cDNA sequence, genomic structure, and general implications for supramolecular assemblies

A Drosophila melanogaster gene for a basement membrane procollagen chain was recently identified from the sequence homology of the carboxyl (NC1) end of the polypeptide that it encodes with the corresponding domain of human and murine collagens IV (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). This gene is at chromosome location 25C. Here we report the complete 6-kilobase cDNA sequence coding for a chain of 1775 amino acids, as well as the genomic structure. The gene is composed of nine relatively large exons separated by eight relatively small introns. This organization is different from the multiple small exons separated by large introns reported for mouse and human type IV collagens (Kurkinen, M., Bernard, M. P., Barlow, D. P., and Chow, L. T. (1985) Nature 317, 177-179. Sakurai, Y., Sullivan, M., and Yamada, Y. (1986) J. Biol. Chem. 261, 6654-6657. Soininen, R., Tikka, L., Chow, L., Pihlajaniemi, T., Kurkinen, M., Prockop, D. J., Boyd, C. D., and Tryggvason, K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1568-1572). Drosophila and human alpha 1(IV) procollagen chains share not only polypeptide domains near their amino and carboxyl ends for making specialized, intermolecular junctional complexes, but also 11 of 21 sites of imperfections of the collagen triple helix. However, neither the number nor the nature of the amino acids in these imperfections appear to have been conserved. These imperfections of the helical sequence may be important for the supramolecular assembly of basement membrane collagen. The 9 cysteine residues of the Drosophila collagen thread domain are arranged as several variations of a motif found in vertebrate collagens IV only near their amino ends, in their "7 S" junctional domains. The relative positions of these cysteine residues provide numerous opportunities for disulfide bonding between molecules in both parallel and antiparallel arrays. There is a pseudorepeat of one-third of the thread length, and there are numerous possibilities for disulfide-linked microfibrils and networks. We propose that collagen microfibrils, stabilized by disulfide segment junctions, are a versatile ancestral form from which specialized collagen fibers and networks arose.     

Two highly conserved glutamic acid residues in the predicted beta propeller domain of dipeptidyl peptidase IV are required for its enzyme activity

Dipeptidyl peptidase IV (DPP IV) is a member of the prolyl oligopeptidase family and modifies the biological activities of certain chemokines and neuropeptides by cleaving their N-terminal dipeptides. This paper reports the identification and possible significance of a novel conserved sequence motif Asp-Trp-(Val/Ile/Leu)-Tyr-Glu-Glu-Glu (DW(V/I/L)YEEE) in the predicted beta propeller domain of the DPP IV-like gene family. Single amino acid point mutations in this motif identified two glutamates, at positions 205 and 206, as essential for the enzyme activity of human DPP IV. This observation suggests a novel role in proteolysis for residues of DPP IV distant from the Ser-Asp-His catalytic triad.     

Oligomerization,F-actin interaction,and membrane association of the ubiquitous mammalian coronin 3 are mediated by its carboxyl terminus

Coronin 3 is a ubiquitously expressed member of the coronin protein family in mammals. In fibroblasts and HEK 293 cells, it is localized both in the cytosol and in the submembranous cytoskeleton, especially at lamellipodia and membrane ruffles. The carboxyl terminus of all coronins contains a coiled coil suggested to mediate dimerization. We show here that in contrast to other coronin homologues, the recombinant human coronin 3 carboxyl terminus forms oligomers rather than dimers, and that this part is sufficient to bind to and cross-link F-actin in vitro. The carboxyl terminus alone also conferred membrane association in vivo, and removal of the coiled coil abolished membrane localization but not in vitro F-actin binding. Coronin 3 is exclusively extracted as an oligomer from both the cytosol and the membrane fraction. Because oligomerization was not reported for other coronins, it might be a key feature governing coronin 3-specific functions. Cytosolic coronin 3 showed a high degree of phosphorylation, which is likely to regulate the subcellular localization of the protein.     

Coils in the membrane core are conserved and functionally important

With the increasing number of available α-helical transmembrane (TM) protein structures, the traditional picture of membrane proteins has been challenged. For example, reentrant regions, which enter and exit the membrane at the same side, and interface helices, which lie parallel with the membrane in the membrane-water interface, are common. Furthermore, TM helices are frequently kinked, and their length and tilt angle vary. Here, we systematically analyze 7% of all residues within the deep membrane core that are in coil state. These coils can be found in TM-helix kinks as major breaks in TM helices and as parts of reentrant regions.Coil residues are significantly more conserved than other residues. Due to the polar character of the coil backbone, they are either buried or located near aqueous channels. Coil residues are frequently found within channels and transporters, where they introduce the flexibility and polarity required for transport across the membrane. Therefore, we believe that coil residues in the membrane core, while constituting a structural anomaly, are essential for the function of proteins.     

Basement membrane and its components on lymphocyte adhesion, migration, and proliferation.

During inflammation and recirculation, lymphocytes migrate into tissues by traversing the capillary endothelium, a process known as extravasation. After crossing the endothelial cells, lymphocytes come into contact with the basement membrane, which is a specialized layer of extracellular matrix containing predominantly laminin, collagen type IV, entactin, and heparan sulfate proteoglycans. In tissue invasion by inflammatory cells and metastatic tumor cells, the basement membrane serves as a substratum for cell adhesion and migration. However, the role of basement membrane in lymphocyte extravasation remains unclear. In this study, we investigated the effect of basement membrane on lymphocyte adhesion, migration, and proliferation, using matrigel as a model for basement membrane. We observed that matrigel promotes both lymphocyte adhesion and migration, with entactin primarily responsible for promoting adhesion and laminin for promoting migration. In addition, activation of lymphocytes by anti-CD3 enhances their adhesion and migration on matrigel-coated substratum. We also observed that matrigel inhibits the proliferation of lymphocytes stimulated by Con A. Furthermore, we demonstrated that laminin is the matrigel component responsible for inhibiting lymphocyte proliferation. However, matrigel has no effect on the proliferation of lymphocytes stimulated by LPS. These results suggest that matrigel has different effects on lymphocyte subpopulations. In agreement with the results on proliferation, matrigel also inhibits the production of IL-2 by Con A-stimulated lymphocytes.     

Drosophila laminin A chain sequence, interspecies comparison, and domain structure of a major carboxyl portion.

Recent studies ascribed some biological actions of cell adhesion and cell outgrowth to the carboxyl-most 1200 amino acids of vertebrate laminin A chains. Here we report a 6.1-kilobase pair nucleotide cDNA sequence encoding 1951 amino acids and the carboxyl end of a Drosophila laminin A chain. It corresponds to the mouse laminin A domains G, I, II, and III, but may represent a different type of laminin A chain. The arrangement of the cysteine-rich repeats of domain III resembles that of B2 chains. However, it has more amino acid identity with a portion of the mouse laminin A chain domain IIIb than with other laminin repeats. Domains I and II are consistent with an interrupted coiled-coil alpha-helical model of the long arm of laminin but are poorly conserved. The G domain contains five subdomains which are individually related to subdomains of vertebrate laminin A chains. The results indicate that laminin G subdomains should be considered individually, rather than merely as parts of a G-globule. A sequence of hydroxyamino acids contributes to a spacer between two of the subdomains. Stretches of hydroxyamino acids may be indicative of junctions between domains of extracellular Drosophila proteins.     

Pseudopod membrane in TSH-stimulated thyroid cells: a specialized domain in the neighboring apical plasma membrane

Summary Pseudopod formation in response to thyrotropin can be obtained with porcine thyroid cell monolayers attached to floating collagen gels or collagen-coated Millipore filters, a model system that allows free access to ligands and antibodies to the apical plasma membrane. To obtain new insight concerning the molecular composition of the pseudopod membrane, (1) ligands were used allowing identification of anionic sites (ruthenium red, cationized ferritin) or carbohydrate units (wheat germ agglutinin, WGA) and (2) antibodies elicited against isolated porcine thyroid membranes or dog intestinal aminopeptidase were employed.Wheat germ agglutinin-binding sites, detected by fluorescence and electron microscopy, were heterogeneously dispersed on the apical membrane. In TSH-stimulated cells, the absence of WGA-binding sites was showed on the pseudopod membrane of thyroid cells, in addition to the previously reported absence of anionic sites. This absence of binding appeared to be independent of the conditions of incubation and/or times of stimulation. Aminopeptidase, which is an apical marker in thyroid cells, was redistributed and clustered on the pseudopod membrane in the cells exposed to TSH stimulation.These present findings support the view that the pseudopod surface constitutes a highly specialized microdomain within the thyroid apical plasma membrane during TSH acute stimulation.With the technical assistance of Brigitte Nguyen Than Dao, Laboratoire de Neuroendocrinologie A, U.S.T.L., Montpellier. Preliminary accounts of this study were presented at the XXI-Vème Colloque de la Société Française de Biologie Cellulaire, Montpellier, 1984     

Rat prominin, like its mouse and human orthologues, is a pentaspan membrane glycoprotein

Mouse prominin is the first characterized member of a novel family of membrane glycoproteins. It displays a characteristic membrane topology with five transmembrane segments and two large glycosylated extracellular loops. Prominin orthologues and paralogues have been identified in human, fish, fly, and worm. Recently, a cDNA sequence encoding the rat homologue of mouse prominin has been reported [Zhu et al. (2001) Biochem. Biophys. Res. Commun. 281, 951-956]. Surprisingly, due to a single nucleotide deletion that shifts the reading frame and introduces a premature stop codon, the protein predicted from this cDNA would correspond to a C-terminally truncated form of prominin with only four transmembrane segments. Here we report evidence that is in contrast to the report of Zhu et al. (2001). We isolated a rat prominin cDNA devoid of any frameshift mutation, demonstrate that rat prominin, like the other mammalian prominins, is a full-length 120-kDa pentaspan membrane glycoprotein, and have not been able to detect any C-terminally truncated form of rat prominin.     

Minor pilin subunits are conserved in Vibrio cholerae type IV pili

The nucleotide sequences of five open reading frames within the Vibrio cholerae NAGV14 type IV pilus gene cluster were determined. The genes showed high homology to the mannose-sensitive hemagglutinin (MSHA) pilus genes mshB, mshC, mshD, mshO and mshP. PCR analysis showed that a MSHA-like gene cluster is highly conserved among different V. cholerae strains, with the exception of the previously reported major pilin subunit. Recombinant MshB and MshO proteins were purified and specific antiserum was raised to each of them. Western blotting analyses showed that these antisera reacted with purified NAGV14 and MSHA pili. The results suggested that MshB and MshO are minor components of the pilus fiber. Although there was no cross-reaction between the major pilin subunits of NAGV14 and MSHA pili, minor components seemed to be highly homologous and immunologically cross-reactive.     

The processing of procollagen in cultures of human and mouse fibroblasts.

Cultures of normal human and mouse fibroblasts convert procollagen to tropocollagen at varying rates. The conversion rate cannot be predicted from the species of origin of the fibroblast nor from the age of the donor tissue. Procollagen is converted to tropocollagen in both the extracellular space of the cell layer and in the culture medium. The collagen fibers of the cell layer are formed mostly from tropocollagen molecules generated in situ.     

Biosynthesis of two subunits of type IV procollagen and of other basement membrane proteins by a human tumor cell line

The major collagenous component secreted into the medium of cultured HT-1080 tumor cells was identified as type IV procollagen by specific antibodies and characteristic ratios of incorporated labeled 3-hydroxyproline and 4-hydroxyproline. The disulfide-bonded molecules consisted of two subunits, pro-alpha 1(IV) and pro-alpha 2(IV) chains with apparent molecular weights of 180 000 and 165 000. No conversion of the procollagen to collagen or to procollagen intermediates was detected in the cell cultures. The two subunits apparently represent different gene products, since enzymatic digestion of the separated chains produced quite different peptide maps. Pepsin degraded native type IV procollagen successively into several fragments, some still disulfide-linked, giving rise to a complex set of polypeptide chains (Mr = 30 000-140 000). This agrees with similar diverse patterns produced by pepsin from authentic type IV collagens. The ratio between the pro-alpha 1(IV) and pro-alpha 2(IV) chains varied in several experiments between 1.3 and 1.8, suggesting that the two chains belong to different triple-helical molecules. The cells also produced distinct amounts of fibronectin (subunit Mr = 230 000) and of the basement membrane glycoprotein laminin. The latter showed three subunits with Mr = 220 000, 210 000, and 400 000. A further disulfide-bonded, non-collagenous polypeptide (Mr = 160 000) was detected but not yet identified. Immunofluorescence demonstrated these proteins within the cells but not in a pericellular matrix. The production of basement membrane components by HT-1080 cells and lack of interstitial collagens disagree with the original classification of the cell line as a fibrosarcoma.     

Multimerization of the mouse TATA-binding protein (TBP) driven by its C-terminal conserved domain.

The conformational states of the mouse TATA-binding protein (TBP) in solution were studied. A histidine tag and a factor Xa recognition site-carrying mouse TBP was expressed in E. coli, highly purified, and its fundamental functions as a TBP were demonstrated. We analyzed the molecular states of mouse TBP by gel filtration and glycerol gradient sedimentation, and found that TBP forms heterogeneous multimers in solution. Direct binding of TBP molecules to each other was proven by the far-Western procedure. Analyses using TBPs truncated at the N- and C-termini demonstrated that the functionally important C-terminal domain was responsible for homomultimer formation, and the N-terminal domain enhances multimerization. Furthermore, it was found that the TATA sequence dissociates homomultimers, and only monomeric TBP binds to the TATA-box. We suggest that TBP shares structural motifs in the C-terminal conserved domain for intermolecular interaction and TATA-binding.     

Identification of a mouse germ cell-less homologue with conserved activity in Drosophila

Drosophila Germ cell-less (Gcl) has previously been shown to be important in early events during the formation of pole cells, which are the germ cell precursors in the fly. We have isolated a 524 amino acid mouse gene with 32% identity and 49% similarity to Drosophila gcl, termed mgcl-1. Like Drosophila Gcl, mGcl-1 localizes to the nuclear envelope. Ectopic expression of mgcl-1 in Drosophila rescues the gcl-null phenotype, indicating that mGcl-1 is a functional homologue of Gcl. mgcl-1 maps to chromosome 6 at 47.3 cM, and is expressed at low levels at all embryonic stages examined from 8.5 to 18.5 d.p.c. as well as in many adult tissues. Different from Drosophila gcl, mgcl-1 is not highly expressed at the time the primordial germ cells appear in the mouse, but high mgcl-1 expression is found in selected mouse adult male germ cells. The differences in these expression patterns in light of conserved activity between the two genes is discussed.     

Two conserved residues are important for inducing highly ordered membrane domains by the transmembrane domain of influenza hemagglutinin

The interaction with lipids of a synthetic peptide corresponding to the transmembrane domain of influenza hemagglutinin was investigated by means of electron spin resonance. A detailed analysis of the electron spin resonance spectra from spin-labeled phospholipids revealed that the major effect of the peptide on the dynamic membrane structure is to induce highly ordered membrane domains that are associated with electrostatic interactions between the peptide and negatively charged lipids. Two highly conserved residues in the peptide were identified as being important for the membrane ordering effect. Aggregation of large unilamellar vesicles induced by the peptide was also found to be correlated with the membrane ordering effect of the peptide, indicating that an increase in membrane ordering, i.e., membrane dehydration, is important for vesicle aggregation. The possibility that hydrophobic interaction between the highly ordered membrane domains plays a role in vesicle aggregation and viral fusion is discussed.