Molecular cloning of syndecan, an integral membrane proteoglycan

We describe cDNA clones for a cell surface proteoglycan that bears both heparan sulfate and chondroitin sulfate and that links the cytoskeleton to the interstitial matrix. The cDNA encodes a unique core protein of 32,868 D that contains several structural features consistent with its role as a glycosamino-glycan-containing matrix anchor. The sequence shows discrete cytoplasmic, transmembrane, and NH2-terminal extracellular domains, indicating that the molecule is a type I integral membrane protein. The cytoplasmic domain is small and similar in size but not in sequence to that of the beta-chain of various integrins. The extracellular domain contains a single dibasic sequence adjacent to the extracellular face of the transmembrane domain, potentially serving as the protease-susceptible site involved in release of this domain from the cell surface. The extracellular domain contains two distinct types of putative glycosaminoglycan attachment sites; one type shows sequence characteristics of the sites previously described for chondroitin sulfate attachment (Bourdon, M. A., T. Krusius, S. Campbell, N. B. Schwartz, and E. Ruoslahti. 1987. Proc. Natl. Acad. Sci. USA. 84:3194-3198), but the other type has newly identified sequence characteristics that potentially correspond to heparan sulfate attachment sites. The single N-linked sugar recognition sequence is within the putative chondroitin sulfate attachment sequence, suggesting asparagine glycosylation as a mechanism for regulating chondroitin sulfate chain addition. Both 5' and 3' regions of this cDNA have sequences substantially identical to analogous regions of the human insulin receptor cDNA: a 99-bp region spanning the 5' untranslated and initial coding sequences is 67% identical and a 35-bp region in the 3' untranslated region is 81% identical in sequence. mRNA expression is tissue specific; various epithelial tissues show the same two sizes of mRNA (2.6 and 3.4 kb); in the same relative abundance (3:1), the cerebrum shows a single 4.5-kb mRNA. This core protein cDNA describes a new class of molecule, an integral membrane proteoglycan, that we propose to name syndecan (from the Greek syndein, to bind together).     

Development of needle retention in Scots pine (Pinus sylvestris) in 1957–1991 in northern and southern Finland

The needle trace method was used to study retrospectively the long-term latitudinal variation in needle retention in Scots pine (Pinus sylvestris L.) in Finland. The mean annual summer needle retention (ANR) along the main stem varied from 3.4 to 6.0 needle sets during the period 1957–1991. The lowest values were observed in southern and the highest in northern Finland. The length of the growing season, expressed as the thermal sum (threshold value +5 °C), was negatively correlated with the mean ANR (r=-0.96). The geographical needle retention pattern (NRP) for the period 1957–1991 showed a clearly increasing trend from 1957 to 1969 (southern Finland) and to 1975 (northern Finland); thereafter, the NRP tended to decrease close to its minimum value recorded in 1991. The general level of the NRP was approximately 5.0 needle sets in northern Finland and 3.5–4.0 needle sets in southern Finland. The NRP, with its 6–12 year cycle for southern Finland, was clearly periodical. Differences in the NRP among the ten stands in southern Finland were small, whereas the said periodicity was missing and the differences were high among the stands in northern Finland. The results indicate that variation in the number of needle sets, viz. defoliation of pines, is a normal phenomenon. The role of net carbon assimilation as a regulator of the number of needle sets is discussed.     

Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin.

The lymphocyte-high endothelial venule (HEV) cell interaction is an essential element of the immune system, as it controls lymphocyte recirculation between blood and lymphoid organs in the body. This interaction involves an 85-95-kD class of lymphocyte surface glycoprotein(s), CD44. A subset of lymphocyte CD44 molecules is modified by covalent linkage to chondroitin sulfate (Jalkanen, S., M. Jalkanen, R. Bargatze, M. Tammi, and E. C. Butcher. 1988. J. Immunol. 141:1615-1623). In this work, we show that removal of chondroitin sulfate by chondroitinase treatment of lymphocytes or incubation of HEV with chondroitin sulfate does not significantly inhibit lymphocyte binding to HEV, suggesting that chondroitin sulfate is not involved in endothelial cell recognition of lymphocytes. Affinity-purified CD44 antigen was, on the other hand, observed to bind native Type I collagen fibrils, laminin, and fibronectin, but not gelatin. Binding to fibronectin was studied more closely, and it was found to be mediated through the chondroitin sulfate-containing form of the molecule. The binding site on fibronectin was the COOH-terminal heparin binding domain, because (a) the COOH-terminal heparin-binding fragment of fibronectin-bound isolated CD44 antigen; (b) chondroitin sulfate inhibited this binding; and (c) finally, the ectodomain of another cell surface proteoglycan, syndecan, which is known to bind the COOH-terminal heparin binding domain of fibronectin (Saunders, S., and M. Bernfield. 1988. J. Cell Biol. 106: 423-430), inhibited binding of CD44 both to intact fibronectin and to its heparin binding domain. Moreover, inhibition studies showed that binding of a lymphoblastoid cell line, KCA, to heparin binding peptides from COOH-terminal heparin binding fragment of fibronectin was mediated via CD44. These findings suggest that recirculating lymphocytes use the CD44 class of molecules not only for binding to HEV at the site of lymphocyte entry to lymphoid organs as reported earlier but also within the lymphatic tissue where CD44, especially the subset modified by chondroitin sulfate, is used for interaction with extracellular matrix molecules such as fibronectin.     

Binding of human syndecan to extracellular matrix proteins.

We have isolated a cell surface proteoglycan from a human mammary cell line (HBL-100). This proteoglycan was found to be a human equivalent to mouse syndecan, because (i) it has identical biochemical properties with murine syndecan, including size, charge, buoyant density, and glycosaminoglycan composition, (ii) its core protein has identical size with murine syndecan as studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iii) the core protein is detected with anti-peptide antibody for the cytoplasmic domain of syndecan. HBL-100 cells also showed high expression of syndecan mRNAs, when probed with mouse syndecan cDNA. The ectodomain of the human syndecan revealed binding to type I collagen fibrils and fibronectin but not to laminin, duplicating the binding properties of murine syndecan. Very interestingly, syndecan did not bind to vitronectin, which is known to contain a heparin binding domain and is one of the major adhesive factors of serum for cultured cells. Syndecans are known to change their glycosaminoglycan composition yielding tissue-type specific polymorphic forms of syndecan (Sanderson, R., and Bernfield, M. (1988) Proc. Natl. Acad. Sci. U. S.A. 85, 9562-9566). The members of this family may thus represent a collection of structurally related matrix receptors that could differ in their interactions due to variation of the ectodomain glycosylation.     

Characterisation of a novel Emaravirus identified in mosaic-diseased Eurasian aspen (Populus tremula)

Since Emaraviruses have been discovered in 2007 several new species were detected in a range of host plants. Five genome segments of a novel Emaravirus from mosaic-diseased Eurasian aspen (Populus tremula) have been completely determined. The monocistronic, segmented ssRNA genome of the virus shows a genome organisation typical for Emaraviruses encoding the viral RNA-dependent RNA polymerase (RdRP, 268.2 kDa) on RNA1 (7.1 kb), a glycoprotein precursor (GPP, 73.5 kDa) on RNA2 (2.3 kb), the viral nucleocapsid protein (N, 35.6 kDa) on RNA3 (1.6 kb), and a putative movement protein (MP, 41.0 kDa) on RNA4 (1.6 kb). The fifth identified genome segment (RNA5, 1.3 kb) encodes a protein of unknown function (P28, 28.1 kDa). We discovered that it is distantly related to proteins encoded by Emaraviruses, such as P4 of European mountain ash ringspot-associated virus. All proteins from this group contain a central hydrophobic region with a conserved secondary structure and a hydrophobic amino acid stretch, bordered by two highly conserved positions, thus clearly representing a new group of homologues of Emaraviruses. The virus identified in Eurasian aspen is closely associated with observed leaf symptoms, such as mottle, yellow blotching, variegation and chloroses along veins. All five viral RNAs were regularly detectable by RT-PCR in mosaic-diseased P. tremula in Norway, Finland and Sweden (Fennoscandia). Observed symptoms and testing of mosaic-diseased Eurasian aspen by virus-specific RT-PCR targeting RNA3 and RNA4 confirmed a wide geographic distribution of the virus in Fennoscandia. We could demonstrate that the mosaic-disease is graft-transmissible and confirmed that the virus is the causal agent by detection in symptomatic, graft-inoculated seedlings used as rootstocks as well as in the virus-infected scions used for graft-inoculation. Owing to these characteristics, the virus represents a novel species within the genus Emaravirus and was tentatively denominated aspen mosaic-associated virus.     

Characterization of the D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate

The murine gene for the glucuronyl C5-epimerase involved in heparan sulfate biosynthesis was cloned, using a previously isolated bovine lung cDNA fragment (Li, J.-P., Hagner-McWhirter, A., Kjellén, L., Palgi, J., Jalkanen, M., and Lindahl, U. (1997) J. Biol. Chem. 272, 28158-28163) as probe. The approximately 11-kilobase pair mouse gene contains 3 exons from the first ATG to stop codon and is localized to chromosome 9. Southern analysis of the genomic DNA and chromosome mapping suggested the occurrence of a single epimerase gene. Based on the genomic sequence, a mouse liver cDNA was isolated that encodes a 618-amino acid residue protein, thus extending by 174 N-terminal residues the sequence deduced from the (incomplete) bovine cDNA. Comparison of murine, bovine, and human epimerase cDNA structures indicated 96-99% identity at the amino acid level. A cDNA identical to the mouse liver species was demonstrated in mouse mast cells committed to heparin biosynthesis. These findings suggest that the iduronic acid residues in heparin and heparan sulfate, despite different structural contexts, are generated by the same C5-epimerase enzyme. The catalytic activity of the recombinant full-length mouse liver epimerase, expressed in insect cells, was found to be >2 orders of magnitude higher than that of the previously cloned, smaller bovine recombinant protein. The approximately 52-kDa, similarly highly active, enzyme originally purified from bovine liver (Campbell, P., Hannesson, H. H., Sandb?ck, D., Rodén, L., Lindahl, U., and Li, J.-P. (1994) J. Biol. Chem. 269, 26953-26958) was found to be associated with an approximately 22-kDa peptide generated by a single proteolytic cleavage of the full-sized protein.     

Expression and glycosylation studies of human FGF receptor 4

Fibroblast growth factor receptor subtype 4 (FGFR4) has been shown to have special activation properties and just one splicing form, unlike the other FGFRs. FGFR4 overexpression is correlated with breast cancer and therefore FGFR4 is a target for drug design. Our aim is to overexpress high amounts of homogeneous FGFR4 extracellular domain (FGFR4(ed)) for structural studies. We show that baculovirus-insect cell-expressed FGFR4(ed) is glycosylated on three (N88, N234, and N266) of the six possible N-glycosylation sites but is not O-glycosylated. The deglycosylated triple mutant was expressed and had binding properties similar to those of glycosylated FGFR4(ed), but was still heterogeneous. Large amounts of FGFR4(ed) have been produced into inclusion bodies in Escherichia coli and refolded at least partly correctly but the refolded E. coli-produced FGFR4(ed) still aggregates.     

Cell surface monoamine oxidases: enzymes in search of a function

Ectoenzymes with a catalytically active domain outside the cell surface have the potential to regulate multiple biological processes. A distinct class of copper-containing semicarbazide-sensitive monoamine oxidases, expressed on the cell surface and in soluble forms, oxidatively deaminate primary amines. Via transient covalent enzyme-substrate intermediates, this reaction results in production of aldehydes, hydrogen peroxide and ammonium, which are all biologically active substances. The physiological functions of these enzymes have remained unknown, although they have been suggested to be involved in the metabolism of biogenic amines. Recently, new roles have been proposed for these enzymes in regulation of glucose uptake and, even more surprisingly, in leukocyte-endothelial cell interactions. The emerging functions of ectoenzymes in signalling and cell-cell adhesion suggest a novel mode of molecular control of these complex processes.     

Primary Amine Oxidase of Escherichia coli Is a Metabolic Enzyme that Can Use a Human Leukocyte Molecule as a Substrate

Escherichia coli amine oxidase (ECAO), encoded by the tynA gene, catalyzes the oxidative deamination of aromatic amines into aldehydes through a well-established mechanism, but its exact biological role is unknown. We investigated the role of ECAO by screening environmental and human isolates for tynA and characterizing a tynA-deletion strain using microarray analysis and biochemical studies. The presence of tynA did not correlate with pathogenicity. In tynA+ Escherichia coli strains, ECAO enabled bacterial growth in phenylethylamine, and the resultant H₂O₂ was released into the growth medium. Some aminoglycoside antibiotics inhibited the enzymatic activity of ECAO, which could affect the growth of tynA+ bacteria. Our results suggest that tynA is a reserve gene used under stringent environmental conditions in which ECAO may, due to its production of H₂O₂, provide a growth advantage over other bacteria that are unable to manage high levels of this oxidant. In addition, ECAO, which resembles the human homolog hAOC3, is able to process an unknown substrate on human leukocytes.     

The VA, VCD, Raman and ROA spectra of tri-L-serine in aqueous solution

The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory with the Becke 3LYP hybrid exchange correlation (XC) functional and the PW91 GGA XC functional and the 6-31G* and aug-cc-pVDZ basis sets. Subsequently, the vibrational absorption, vibrational circular dichroism, Raman and Raman optical activity spectra were simulated in order to compare them to experimentally measured spectra. In addition, we compare to previously reported studies for both structural determination and spectral simulations and measurements. A comparison of the various ways to treat the effects of the environment and solvation on both the structure and the spectral properties is thoroughly investigated for one conformer, with the goal to determine which level of theory is appropriate to use in the systematic search of the conformational space. In addition, the effects of the counterion, here Cl- anion, are also investigated. Here we present the current state of the art in nanobiology, where the latest methods in experimental and theoretical vibrational spectroscopy are used to gain useful information about the coupling of the nuclear, electronic and magnetic degrees of freedom and structure of tri-L-serine and its capped peptide analog with the environment.