Second Harmonic Generation of Nanoscale Phonon Wave Packets.

Phonons are often regarded as delocalized quasiparticles with certain energy and momentum. The anharmonic interaction of phonons determines macroscopic properties of the solid, such as thermal expansion or thermal conductivity, and a detailed understanding becomes increasingly important for functional nanostructures. Although phonon-phonon scattering processes depicted in simple wave-vector diagrams are the basis of theories describing these macroscopic phenomena, experiments directly accessing these coupling channels are scarce. We synthesize monochromatic acoustic phonon wave packets with only a few cycles to introduce nonlinear phononics as the acoustic counterpart to nonlinear optics. Control of the wave vector, bandwidth, and consequently spatial extent of the phonon wave packets allows us to observe nonlinear phonon interaction, in particular, second harmonic generation, in real time by wave-vector-sensitive Brillouin scattering with x-rays and optical photons.

Coupling of GHz phonons to ferroelastic domain walls in SrTiO3.

We study the linear and nonlinear acoustic response of SrTiO3 across its ferroelastic transition at Ta=105 K by time domain Brillouin scattering. Above Ta we observe that for a strain amplitude of ∼0.18% the sound velocity for compressive strain exceeds the tensile strain velocity by 3%. Below Ta we find a giant slowing down of the sound velocity by 12% and attribute this to the coupling of GHz phonons to ferroelastic twin domain walls. We propose a new mechanism for this coupling on the ultrafast time scale, providing an important new test ground for theories used to simulate atomic motion in domain forming crystals.

The adhesive properties of recombinant soluble L-selectin are modulated by its glycosylation.

The leukocyte adhesion molecule L-selectin, which mediates the initial steps of leukocyte attachment to vascular endothelium, is intensely glycosylated. Different glycoforms of L-selectin are expressed on different leukocyte subsets and differences in L-selectin glycosylation appear to be correlated with the leukocyte's ability to attach to different endothelial targets. In the present study we addressed the question whether glycosylation of L-selectin influences L-selectin-ligand interactions. To obtain different glycoforms of L-selectin, recombinant proteins were expressed both in the baby hamster kidney (BHK) cell line and in the human myelogenous cell line K562, resulting in sL-sel[BHK] or sL-sel[K562], respectively. The glycosylation characteristics of the purified proteins were determined. The most striking differences in glycosylation were seen in the terminal sialylation. Each of the two proteins carried sialic acids in the alpha 2-3 position, while alpha 2-6-bound sialic acids were found exclusively on sL-sel[K562]. To investigate their adhesive properties, both recombinant sL-selectins were used in cell adhesion assays and interactions with the ligands present on various hematopoietic cell lines or activated human cardiac microvascular endothelial cells were examined. The binding capacity of sL-sel[K562] was about 1.6 fold higher compared to sL-sel[BHK] under static as well as under flow conditions. These findings indicate that the terminal sialylation pattern of L-selectin modulates its binding characteristics.

Carbohydrate structures of soluble human L-selectin recombinantly expressed in baby-hamster kidney cells.

A soluble form of L-selectin was recombinantly produced, which might be an effective therapeutic agent in inflammatory disorders, acting as an inhibitor for leucocyte endothelium adhesion. In the present study the oligosaccharide structures of soluble human L-selectin, recombinantly expressed in baby-hamster kidney cells, were determined. The N-linked glycans were enzymically released and fluorescently labelled with 2-aminobenzamide. Sialylation of the N-glycans was analysed by anion-exchange chromatography followed by rechromatography of the resulting fractions on amino-phase HPLC after release of the sialic acid residues. Desialylated oligosaccharides were separated using two-dimensional HPLC and characterized by digestion with exoglycosidases and MS. More than 30 oligosaccharide structures representing at least 95% of the overall glycosylation of this protein were determined. The results revealed that recombinant soluble human L-selectin carries bi-, tri- and tetra-antennary sugar chains, which are fucosylated on the innermost residue of N-acetylglucosamine. The number of sialic acid residues linked to these glycans ranges from 0 (neutral glycans) to 4 (tetrasialylated oligosaccharides). The sialic acid is found exclusively in the alpha 2-3 linkage to galactose. In addition to the main glycans, different minor structures containing terminal N-acetylgalactosamine, or the H (O) blood-group determinant were also identified. O-Glycosylation of mucin-type sugar chains was not detected in recombinant soluble human L-selectin.

In vivo modulation of the acidic N-glycans from rat liver dipeptidyl peptidase IV by N-propanoyl-D-mannosamine.

Derivatives of N-acyl-D-mannosamine differing in the N-acyl-side chain can be metabolically converted into neuraminic acids with corresponding N-acyl side chains. In the present study we show the in vivo modulation of sialic acids in membrane-bound dipeptidyl peptidase IV (CD 26) from rat liver after administration of N-propanoyl-D-mannosamine. Treatment of rats with this unphysiological precursor resulted in an incorporation of N-propanoylneuraminic acid into N-linked glycans of dipeptidyl peptidase IV.

N-glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures.

Rat C-CAM is a ubiquitous, transmembrane and carcinoembryonic antigen related cell adhesion molecule. The human counterpart is known as biliary glycoprotein (BGP) or CD66a. It is involved in different cellular functions ranging from intercellular adhesion, microbial receptor activity, signaling and tumor suppression. In the present study N-glycosylation of C-CAM immunopurified from rat liver was analyzed in detail. The primary sequence of rat C-CAM contains 15 potential N-glycosylation sites. The N-glycans were enzymatically released from glycopeptides, fluorescently labeled with 2-aminobenzamide, and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by enzymatic sequencing and MALDI-TOF-MS. Mainly bi- and triantennary complex structures were identified. The presence of type I and type II chains in the antennae of these glycans results in heterogeneous glycosylation of C-CAM. Sialylation of the sugars was found to be unusual; bi- and triantennary glycans contained three and four sialic acid residues, respectively, and this linkage seemed to be restricted to the type I chain in the antennae. Approximately 20% of the detected sugars contain these unusual numbers of sialic acids. C-CAM is the first transmembrane protein found to be oversialylated.

H (0) blood group determinant is present on soluble human L-selectin expressed in BHK-cells.

In the present study we show that the H (0) blood group determinant Fuc alpha1-2Gal beta1-4GlcNAc beta1-R is present on N-linked glycans of soluble human L-selectin recombinantly expressed in baby hamster kidney (BHK) cells. The glycans were isolated using complementary HPLC techniques and characterized by a combination of exoglycosidase digestion and mass spectrometry. The linkage of the fucose residues was determined by incubation of the glycans with specific fucosidases. The H blood determinant Fuc alpha1-2Gal beta1-4GlcNAc beta1 was detected for bi-, 2,4 branched tri- and tetraantennary structures. To our knowledge, the proposed oligosaccharide structures represent a new glycosylation motif for recombinant glycoproteins expressed on BHK cells.

Rapid analysis of O-acetylated neuraminic acids by matrix assisted laser desorption/ionization time-of-flight mass spectrometry.

N-Acetylneuraminic acid (a sialic acid) occurs mainly as a terminal substituent of oligosaccharides of glycoconjugates. Derivatives of neuraminic acid occur widely, substituted in the amino and hydroxy side chains, as well in the C-9 carbon skeleton. These derivatives are responsible for specific functions of sialic acids during cell-cell, cell-substrate, or cell-virus interactions. The study of O-acetylated neuraminic acids is difficult, because only small amounts are extractable from natural sources and they are generally unstable to acids and bases. We report a new method for the rapid analysis of O-acetylated neuraminic acids, using a combination of reversed phase HPLC and MALDI-TOF mass spectrometry. A mixture of neuraminic acids from bovine submaxillary gland mucins was analysed, as well as neuraminic acids variously substituted in the amino and hydroxy side chains with acetyl and glycolyl groups, respectively.

Analysis of site-specific N-glycosylation of recombinant Desmodus rotundus salivary plasminogen activator rDSPA alpha 1 expressed in Chinese hamster ovary cells.

The recombinant plasminogen activator (rDSPA alpha 1) from the vampire bat Desmodus rotundus is a promising new thrombolytic agent that exhibits a superior pharmacological profile if compared to tissue-type plasminogen activator (t-PA) or streptokinase. In the present study the structures of the carbohydrate moieties at the two N-glycosylation sites (Asn-117, Asn-362) of rDSPA alpha 1 expressed in Chinese hamster ovary cells were determined. N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F digestion and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by analysis of carbohydrate composition and linkage, by mass spectrometry, and by sequence analysis in which the fluorescently labeled glycans were cleaved with an array of specific exoglycosidases. More than 30 different oligosaccharides were identified. The results revealed that Asn-117 carried a mixture of one high-mannose structure (17% of site-specific glycosylation), three hybrid glycans (26%) and predominantly biantennary complex N-glycans (54%). Glycosylation site Asn-362 was found to comprise complex glycans with biantennary (50%), 2,4- and 2,6-branched triantennary (21%, 11%), and tetraantennary structures (10%), which were fucosylated at the innermost residue of N-acetylglucosamine. Mainly neutral and monosialylated glycans, and smaller quantities of disialylated glycans, were detected at both glycosylation sites. Sialic acid was alpha 2-3 linked to galactose exclusively. As shown in this study the N-glycans attached to Asn-117 of rDSPA alpha 1 are more processed during biosynthesis than the high-mannose structures linked to Asn-117 of t-PA, to which the polypeptide backbone of rDSPA alpha 1 is structurally closely related.

O-linked L-fucose is present in Desmodus rotundus salivary plasminogen activator.

DSPAalpha1 (Desmodus rotundus salivary plasminogen activator), a plasminogen activator from the saliva of the vampire bat Desmodus rotundus, is an effective thrombolytic agent. An unusual type of posttranslational modification, in which L-fucose is O-glycosidically linked to threonine 61 in the epidermal growth factor domain was found for natural DSPAalpha1 and its recombinant form isolated from Chinese hamster ovary cells. In the present study a combination of carbohydrate and amino acid composition analysis, amino acid sequencing, and mass spectrometry revealed that the L-fucose is bound to residues 56-68 of DSPAalpha1. The amino acid sequence of this glycosylation site agreed with the suggested consensus sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys described for other proteins. Anew strategy for the identification of the modified amino acid was established. Direct evidence for the occurrence of fucosyl-threonine was obtained by mass spectrometry after digestion of the glycopeptide with a mixture of peptidases. On the basis of these results, DSPAalpha1 is a suitable model for studying the influence of O-fucosylation on clearance rates, particularly in comparative studies with the identically fucosylated and structurally related tissue plasminogen activator.