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PubMed2003

Characterization of monoclonal antibodies to Marburg virus (strain Musoke) glycoprotein and identification of two protective epitopes.

Monoclonal antibodies (MAbs) reactive with Marburg virus (strain Musoke) were evaluated for both biological activity and specificity. Several of the Marburg virus- (MBGV) specific MAbs reduced the size and/or number of MBGV plaques in vitro. The ability of the MAbs to affect plaque formation in vitro was demonstrated to be specific for the glycoprotein (GP) of the strain of MBGV used for vaccination. Using deletion analysis and peptide mapping, the binding epitopes of several of these neutralizing MAbs were identified. Not unexpectedly, the epitopes were shown to lie in the most hypervariable and highly glycosylated region of MBGV GP. An analysis of the in vivo activity of several MAbs revealed that some antibodies provided substantial but incomplete protection of naive guinea pigs by passive transfer. These data suggest that neutralizing epitopes exist within MBGV GP but that induction of antibodies to these neutralizing epitopes may not be sufficient for protection from lethal infection.

Amino Acid Sequence↗
PubMed1985

Identification and characterization of the principal proteins of the fat-globule membrane from guinea-pig milk.

The milk-fat-globule membrane (MFGM) was isolated from guinea-pig milk and the membrane-associated proteins and glycoproteins characterized by electrophoretic techniques. Major components of the membrane included PAS-I, a sialoglycoprotein of Mr greater than or equal to 200000, the redox enzyme xanthine oxidase and the glycoprotein, butyrophilin. Membrane preparations also contained two other glycoproteins, GP-80 and GP-55, of Mr 80000 and 55000, respectively. Comparison of guinea-pig xanthine oxidase and butyrophilin with proteins from bovine MFGM by peptide mapping procedures, showed that the two proteins in both species were similar, but not identical. GP-55 may also be related to glycoproteins of Mr 45000 and 48000 in the bovine membrane. The integral and peripheral components of guinea-pig MFGM were identified by treating membrane preparations with sodium carbonate solutions at high pH and by partitioning the membrane proteins in solutions of Triton X-114. By these criteria xanthine oxidase and GP-55 appeared to be peripheral components and GP-80 an integral protein of the membrane. PAS-I and butyrophilin displayed hydrophilic properties in Triton X-114 solutions, but could not be removed from membrane preparations with sodium carbonate. Possible reasons for these ambiguous data are discussed. The observed similarity between several of the proteins of guinea-pig and bovine MFGM implies that these proteins may have specific functions related to milk secretion in mammary tissue, e.g. in the budding of milk-fat globules or the exocytosis of milk protein and lactose at the apical surface.

Animals↗
PubMed1996

Mutagenesis of transmembrane domain 11 of P-glycoprotein by alanine scanning.

The biochemical and genetic analyses of P-glycoprotein (P-gp) have indicated that the membrane-associated regions of P-gp play an important role in drug recognition and drug transport. Predicted transmembrane domain 11 (TM11) maps near a major drug binding site revealed by photoaffinity labeling, and mutations in this domain alter the substrate specificity of P-gp. To investigate further the role of TM11 in P-gp function in general, and substrate specificity in particular, each of the 21 residues of TM11 of the P-gp isoform encoded by the mouse mdr3 gene was independently mutated to alanine, or to glycine in the case of endogenous alanines. After transfection and overexpression in Chinese hamster ovary cells, pools of stable transfectants were analyzed for qualitative or quantitative deviations from the profile of resistance to vinblastine, adriamycin, colchicine, and actinomycin D displayed by the wild-type protein. While mutations at eight of the positions had no effect on P-gp function, 13 mutants showed a 2-10-fold reduction of activity against one of the four drugs tested. Although the phenotype of individual mutants was varied, replacements at most mutation-sensitive positions seemed to affect the drug resistance profiles rather than the overall activity of the mutant P-gp. When TM11 was projected in a alpha-helical configuration, the distribution of deleterious and neutral mutations was not random but segregated with a more hydrophobic (mutation-insensitive) face and a more hydrophilic (mutation-sensitive) face of a putative amphipathic helix. The alternate clustering pattern of deleterious vs neutral mutations in TM11 together with the altered drug resistance profile of deleterious mutants suggest that the more hydrophilic face of the TM11 helix may play an important structural or functional role in drug recognition and transport by P-gp. Finally, the conservation of the two residues most sensitive to mutations (Y949 and Y953) in TM11, and in the homologous TM5, of all mammalian P-gps and also in other ABC transporters, suggests that these residues and domains may play an important role in structural as well as mechanistic aspects common to this family of proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

The goodpasture autoantigen. Mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC1 domain.

The Goodpasture (GP) autoantigen has been identified as the alpha3(IV) collagen chain, one of six homologous chains designated alpha1-alpha6 that comprise type IV collagen (Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036). In this study, chimeric proteins were used to map the location of the major conformational, disulfide bond-dependent GP autoepitope(s) that has been previously localized to the noncollagenous (NC1) domain of alpha3(IV) chain. Fourteen alpha1/alpha3 NC1 chimeras were constructed by substituting one or more short sequences of alpha3(IV)NC1 at the corresponding positions in the non-immunoreactive alpha1(IV)NC1 domain and expressed in mammalian cells for proper folding. The interaction between the chimeras and eight GP sera was assessed by both direct and inhibition enzyme-linked immunosorbent assay. Two chimeras, C2 containing residues 17-31 of alpha3(IV)NC1 and C6 containing residues 127-141 of alpha3(IV)NC1, bound autoantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactivity with all GP sera and accounting for 50-90% of the autoantibody reactivity toward alpha3(IV)NC1. The conformational nature of the epitope(s) in the C2 and C6 chimeras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from alpha1- and alpha3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in alpha3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.

Amino Acid Sequence↗
PubMed2004

Magnetocardiographic differences of ventricular repolarization parameters between Wistar rats and guinea pigs.

UNLABELLED: The morphology and range of duration of the action potential (AP) in normal Wistar rat's (WR) myocyte markedly differ from those of guinea pigs (GP), whose plateau (phase 2) duration is longer. Thus a clear-cut T wave can be easy defined in GP but not in WR. Aim of this study was to differentiate magnetocardiographic (MCG) ventricular repolarization (VR) parameters of healthy adult WR and GP. METHODS: 10 female animals (5 Guinea pigs and 5 Wistar rats) were studied with a 36-channel MCG system (sensitivity of 20 fT/square root of Hz1/2) and with one ECG lead, in an unshielded hospital room. To assess VR, HR-corrected, JTpeak, JTend, Tpeak-end and QTend, intervals were measured from both MCG waveforms. Timing was improved by MCG maps analysis. Magnetic field orientation (MFO), its dynamics (MFD) and stability (JTS) during the JT interval, were also automatically computed from MCG maps. RESULTS: All repolarization intervals were significantly shorter in WR than in GP, except the Tpeak-end, which was longer. MFO and MFD also differed. CONCLUSIONS: MCG estimate of VR parameters, in adult WR and GP, is precise enough to evidence breed-related differences, consistent with physiological heterogeneity of duration during phases 2 and 3 of the AP, and with an higher degree of transmural dispersion of repolarization in WR.

Animals↗
PubMed1985

A common major surface antigen on amastigotes and promastigotes of Leishmania species.

Enzymatic surface iodination and biosynthetic labeling with [35S]methionine, combined with immunoprecipitation by sera from patients with different forms of Leishmaniasis revealed a 65,000 Mr glycoprotein as the immunodominant moiety in promastigotes and amastigotes of the nine Leishmania species and isolates examined. Sera from patients with one form of Leishmaniasis recognized this component strongly, not only in the homologous, but also in the heterologous species. In addition to the crossreactivity displayed by immune sera, the 65,000 Mr glycoprotein (gp) common to all Leishmania species presented a characteristic shift to Mr 50,000 when samples were run in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. These results are in agreement with our previous studies (7), where a simple and similar profile was obtained for several geographic isolates of L. donovani, with a major surface glycoprotein of 65,000 Mr displaying the same characteristics described here. The structural similarity of the major 65,000 Mr gp of the six Leishmania species was demonstrated by Cleveland mapping. It is suggested that immunological specificities may be contributed by minor differences in glycosylation of this molecule. In keeping with recent data (13-15), where strong cross protection among different Leishmania species has been obtained by prophylactic immunization with irradiated whole promastigotes, this glycoprotein may be a good candidate for an antigen to be used for immunoprophylaxis of all forms of Leishmaniasis.

Antigens, Protozoan↗
PubMed2005

The structure of the GPIb-filamin A complex.

Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbalpha binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbalpha cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIbalpha binds in a groove formed between the C and D beta strands of FLNa domain 17. The interaction is strikingly similar to that between the beta7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.

Contractile Proteins↗
PubMed1989

Platelet glycoprotein Ib beta is phosphorylated on serine 166 by cyclic AMP-dependent protein kinase.

Platelet responses are inhibited by agents such as prostaglandin E1 that increase the cytoplasmic concentration of cyclic AMP. Inhibition is thought to result from phosphorylation of specific proteins. One protein that becomes phosphorylated is glycoprotein (GP) Ib beta, a component of the GP Ib.IX complex. We have suggested that phosphorylation of GP Ib beta inhibits the collagen-induced polymerization of actin. The aim of the present study was to identify the amino acid(s) in GP Ib beta that is phosphorylated. Purified GP Ib.IX complex was phosphorylated by the catalytic subunit of purified bovine cyclic AMP-dependent protein kinase in the presence of [gamma-32P]ATP. Phosphoamino acid analysis showed that in GP Ib beta, [32P]phosphate was incorporated only into serine and was in a single tryptic peptide. Amino acid sequencing showed that this peptide was from the cytoplasmic domain of GP Ib beta and encompassed residues 161-175. A single serine residue, serine 166, contained the radiolabel. To determine whether the same residue was phosphorylated in intact platelets, GP Ib beta was isolated from 32P-labeled platelets before or after their exposure to prostaglandin E1. In both cases, radiolabel was present in phosphoserine and was in a single tryptic peptide. This peptide was the same as that which was phosphorylated in the purified GP Ib.IX complex, as shown by its identical mobility on two-dimensional tryptic maps, the presence of a positively charged residue in the fourth position, and the presence of the radiolabel in the sixth position of the peptide. This study shows that when cyclic AMP concentrations rise in platelets, the cytoplasmic domain of GP Ib beta is phosphorylated on serine 166, probably by cyclic AMP-dependent protein kinase. We suggest that phosphorylation of this residue may contribute to the inhibitory actions of cyclic AMP by inhibiting collagen-induced polymerization of actin.

Adult↗
PubMed1975

Identification of a precursor protein to the major glycoproteins of mouse mammary tumor virus.

Mouse mammary tumor virus-producing cultures of mouse mammary tumor cells synthesize a viral-related polypeptide of molecular weight of 73,000 (gp 73) which is rapidly labeled during a short pulse but disappears during the chase concomitantly with the appearance of label in the virion glycoproteins gp 49 and gp 37.5/33.5. The addition of the protein synthesis-inhibitor cycloheximide to the chase medium has little effect on this conversion. Treatment of the proposed precursor with alpha-chymotrypsin leads to the formation of a polypeptide of molecular weight 49,000, similar to the major virion glycoprotein. A comparison of tryptic digest maps of the glycoproteins involved supports the hypothesis that both the viral glycoproteins gp 49 and gp 37.5/33.5 are derived from gp 73.

Animals↗
PubMedDate not supplied

Radioisotopic lymphatic mapping of the sentinel node in melanoma: importance of immunohistochemistry.

BACKGROUND: Elective lymph node dissection (ELND) for patients with malignant melanoma is still controversial. A possible alternative could be biopsy of the first tumor draining lymph node, the sentinel node (SN), which can be identified by means of radionuclide techniques. AIM: Our study was undertaken to assess the accuracy of lymph node biopsy and to stress the importance of immunohistochemistry (IHC) in the pathological assessment of the SN for improved staging of the primary tumor. METHODS: We performed lymphoscintigraphy (LS) in 183 melanoma patients (89 with melanoma of the legs, 11 of the arms and 83 of the trunk). Our protocol consisted of preoperative peritumoral i.d. injection of 99mTc-labeled microcolloid to define the regional lymphatic basin and identify the sentinel node by means of planar scintigraphy. In 147 of the 183 cases a gamma probe (GP) was used during surgery to trace the SN. Vital blue dye was used during surgery in all cases. The SNs were excised for pathological examination. The pathological status of the SN was defined by means of examination of frozen sections, hematoxylin-eosin staining and immunohistochemistry for S-100 and HMB-45 MAb. RESULTS: At least one separate focus of activity was identified by LS in 182 out of 183 patients; in all 147 cases where a GP was used, it was successful in tracing the SN. LS with cutaneous mapping of the SN successfully guided the surgical excision in 177 of the 183 cases; in the 7 remaining cases, i.e. 7 out of 83 cases with SNs in the axillary basin, GP was not used and no elective node dissection was performed. Metastases were found in 39 of these 177 cases. In all 39 cases the SNs were the only positive nodes in the basin. Of the 39 metastases 18 were identified by means of frozen section, 12 by means of hematoxylin-eosin, and 9 by means of immunohistochemistry. We therefore emphasize the importance of immunohistochemistry in the pathology of LS for improved staging of the primary tumor.

Coloring Agents↗
PubMed1991

Post-translational modifications and binding properties of the apically secreted 80-kDa glycoprotein from Madin-Darby canine kidney cells: similarities to the C-terminal portion of the basolaterally secreted fibronectin.

Apically secreted 80-kDa glycoprotein (gp 80) from Madin-Darby canine kidney cells was found to be immunoprecipitated by the polyclonal antiserum against fibronectin or a monoclonal antibody specific for the fibronectin C-terminal fibrin binding domain. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gp 80 migrated as a doublet band under nonreducing conditions. Under reducing conditions, gp 80 was resolved into three distinct bands, respectively of 45-, 40-, and 35-kDa molecular mass. Analysis by two-dimensional SDS-PAGE revealed that gp 80 exists in two molecular forms: one consisting of a 45-kDa subunit and a 40-kDa subunit, and one consisting of a 45-kDa subunit and a 35-kDa subunit. V-8 protease mapping indicated the 40 and 35-kDa subunits as being of the same homologous group and also as bearing partial homology to the 45-kDa subunit. Radioactive labeling revealed that labeled gp 80 was subjected to covalent modifications by sulfation and phosphorylation. Sulfate analysis showed that [35S]sulfate-labeled gp 80 contained ca. 2.45 +/- 0.07% tyrosine-bound [35S]sulfate with the rest being presumably carbohydrate-bound. [32P]-Phosphate-labeled gp 80, on the other hand, was found to contain serine-O-phosphate as the predominant phosphorylated amino acid residue. Employing the affinity gel fractionation technique, it was shown that gp 80 exhibited binding affinities toward heparin and fibrin. Binding of gp 80 to heparin-agarose or fibrin-Sepharose, however, was inhibited in the presence of added fibronectin or the monoclonal antibody. Tryptic peptide mapping revealed common peptide spots between fibronectin and the three subunits of gp 80. Furthermore, Western blot analysis showed that fibronectin could be recognized and bound by anti-gp 80 antibodies. These results indicate that gp 80 bears both structural and functional similarities to the C-terminal portion of the fibronectin molecule.

Animals↗
PubMed2006

What employees with diabetes mellitus need to cope at work: views of employees and health professionals.

OBJECTIVE: To identify and compare patient and professional perspectives on what enables employees with diabetes mellitus (DM) to maintain their position in the workplace. To provide information on how professionals can help DM patients cope at work. METHODS: Qualitative study using concept-mapping sessions involving 23 employees with DM and 22 health professionals (GP's, occupational physicians and specialists). All of the health professionals were experts in the field of diabetes care. RESULTS: Patients and professionals identified five common clusters of statements on what diabetics need to enable them to cope at work: the ability to accept and cope with DM, supportive health professionals, a supportive work environment, work adaptations and good information. Patients emphasized the importance of emotional acceptance of DM and communication with colleagues, while the professionals emphasized the patient's capacity for self-care. CONCLUSION: The content of patient and professional perspectives on what is needed to prevent work-related problems for DM patients differed slightly. Patients rely on direct experiences in their own environment, professionals on medical knowledge accumulated in groups of patients. PRACTICE IMPLICATIONS: Both perspectives were used to suggest a topic list for health professionals, which may help identify and address the occupational problems experienced by DM patients.

Adaptation, Psychological↗
PubMed2000

Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury.

Here we defined the main viral determinant of Ebola virus pathogenicity; synthesis of the virion glycoprotein (GP) of Ebola virus Zaire induced cytotoxic effects in human endothelial cells in vitro and in vivo. This effect mapped to a serine-threonine-rich, mucin-like domain of this type I transmembrane glycoprotein, one of seven gene products of the virus. Gene transfer of GP into explanted human or porcine blood vessels caused massive endothelial cell loss within 48 hours that led to a substantial increase in vascular permeability. Deletion of the mucin-like region of GP abolished these effects without affecting protein expression or function. GP derived from the Reston strain of virus, which causes disease in nonhuman primates but not in man, did not disrupt the vasculature of human blood vessels. In contrast, the Zaire GP induced endothelial cell disruption and cytotoxicity in both nonhuman primate and human blood vessels, and the mucin domain was required for this effect. These findings indicate that GP, through its mucin domain, is the viral determinant of Ebola pathogenicity and likely contributes to hemorrhage during infection.

Animals↗
PubMed1988

Fine mapping of a peptide sequence containing an antigenic site conserved among arenaviruses.

The lymphocytic choriomeningitis virus (LCMV) structural glycoproteins GP-1 (Mr 44K) and GP-2 (Mr 35K) are encoded on a single intracellular proteolytic cleavage precursor glycoprotein, GP-C (Mr 76K). We have used a series of synthetic peptides derived from the deduced amino acid sequence of LCMV GP-C to define an antigenic site containing two topographically overlapping epitopes. Three mouse monoclonal antibodies directed against two epitopes on GP-2 were assayed for binding in solution phase blocking and solid-phase enzyme-linked immunoadsorbant assays to a series of peptides representing the sequence of the intracellular precursor glycopeptide GP-C. Both epitopes were initially localized to a single peptide GP-C 370-382 (Cys-Asn-Tyr-Ser-Lys-Phe-Trp-Tyr-Leu-Glu-His-Ala-Lys) in the GP-2 segment of GP-C. Further analysis demonstrated that both epitopes were contained within a nine amino acid segment, GP-C 370-378, which contains five residues conserved among LCMV, Lassa, Pichinde, and Tacaribe viruses. Assays with N-terminal deletions from this sequence suggested that the minimal epitope recognized by the broadly cross-reactive monoclonal 33.6 (epitope GP-2a) consisted of five amino acids, GP-C 374-378 (Lys-Phe-Trp-Tyr-Leu). Reactivity of a second monoclonal, 9-7.9 (epitope GP-2B) but not 33.6, was abolished when substitution of tyrosine for phenylalanine was made at position 375 in the antigenic sequence corresponding to a naturally occurring sequence difference between LCM and Lassa viruses. Polyclonal sera from human cases and from animals experimentally infected with Junin, LCM, and Lassa viruses, respectively, bound to the antigenic peptide GP-C 370-382 but not to control peptides. As was the case with the monoclonals, this binding activity was abrogated by blocking with the antigenic peptide but not with control peptides in solution.

Amino Acid Sequence↗
PubMed1992

The genomes of simian varicella virus and varicella zoster virus are colinear.

Simian varicella virus (SVV) causes an exanthematous disease in non-human primates which is clinically similar to varicella zoster virus (VZV) infection of humans. In this study, the genetic relatedness of SVV and VZV was confirmed and the location of SVV DNA sequences homologous to VZV restriction endonuclease (RE) fragments and viral genes was determined. VZV DNA RE fragments representing 98.3% of the VZV genome were 32P-labeled and hybridized to RE digested, immobilized SVV DNA. Homologous sequences were located throughout the viral DNAs in similar map positions, indicating a colinear relationship between the VZV and SVV genomes. 32P-labeled VZV glycoprotein (gp I, II, III, and IV) and gene 62 DNA probes also hybridized to SVV DNA in a colinear manner. The results suggest that the location of specific SVV genes may be predicted from the known map positions of homologous VZV genes. This study provides further support for SVV infection of non-human primates as a model for VZV infection of humans.

Animals↗
PubMed1990

B cell epitope mapping of human immunodeficiency virus envelope glycoproteins with long (19- to 36-residue) synthetic peptides.

Envelope glycoproteins, gp 120 and gp41, of the human immunodeficiency virus type 1 (HIV-1) elicit immune responses, including virus-neutralizing antibodies, which are expected to play a role in the defence against HIV-1 infection. Subregions of the gp120/gp41 sequence have immunosuppressive effects or may be implicated in autoimmune responses. Some of the immunodominant epitopes of gp120/gp41 do not contribute to protective immunity and act as immunological decoys. These circumstances emphasize the need to select from gp120/gp41 regions inducing protective responses. Towards this goal, 30 peptides covering approximately 87% of the HIV-1 strain BH10 gp120/gp41 sequence were synthesized. Antibodies in rabbit and human anti-HIV-1 sera recognized 28 and nine of the peptides, respectively, indicating that most of the gp120/gp41 sequence is immunogenic and secondly, that the antibody response to HIV-1 is restricted in infected humans. Most of the peptides, without conjugation to carriers, elicited high levels of anti-peptide (endpoints 1: greater than 10(4] and anti-gp120/gp41 (endpoints 1: greater than or equal to 10(3] antibodies. The highest levels of virus-neutralizing antibodies were elicited by peptide 306 to 338 from a hypervariable loop of gp120. Additional peptides from the full-length hypervariable loop (303 to 338) of HIV-1 BH10 and from 20 additional HIV-1 isolates were recognized differentially by human anti-HIV, suggesting that success of passive immunization may depend on a match between administered antibodies and the challenging HIV-1 strain, and also that active immunization with selected peptides from a hypervariable region of distinct HIV-1 isolates should be explored further as a method for prophylaxis against infection.

Antibody Specificity↗
PubMed1999

Isoforms of the Lutheran/basal cell adhesion molecule glycoprotein are differentially delivered in polarized epithelial cells. Mapping of the basolateral sorting signal to a cytoplasmic di-leucine motif.

Lu and Lu(v13) are two glycoprotein (gp) isoforms that belong to the immunoglobulin superfamily and carry both the Lutheran (Lu) blood group antigens and the basal cell adhesion molecule epithelial cancer antigen. Lu (85 kDa) and Lu(v13) (78 kDa) gps, which differ only in the length of their cytoplasmic domain, are adhesion molecules that bind laminin. In nonerythroid tissues, the Lu/basal cell adhesion molecule antigens are predominantly expressed in the endothelium of blood vessel walls and in the basement membrane region of normal epithelial cells, whereas they exhibit a nonpolarized expression in some epithelial cancers. Here, we analyzed the polarization of Lu and Lu(v13) gps in epithelial cells by confocal microscopy and domain-selective biotinylation assays. Differentiated human colon carcinoma Caco-2 cells exhibited a polarized expression of endogenous Lu antigens associated with a predominant expression of the Lu isoform at the basolateral domain of the plasma membrane and a very low expression of the Lu(v13) isoform at both the apical and basolateral domains. Analysis of transfected Madin-Darby canine kidney cells revealed a basolateral expression of Lu gp and a nonpolarized expression of Lu(v13) gp. Delivery of Lu(v13) to both apical and basolateral surfaces showed similar kinetics, indicating that this isoform is directly transported to each surface domain. A dileucine motif at position 608-609, specific to the Lu isoform, was characterized as a dominant basolateral sorting signal that prevents Lu gp from taking the apical delivery pathway.

Amino Acid Sequence↗
PubMed1989

Molecular cloning, expression and assembly of multimeric von Willebrand factor.

Recently, substantial progress has been made in our knowledge of the domains involved in correlating structure and function of vWF, as well as in the biosynthesis and assembly of multimeric vWF. These studies were greatly supported by the development of three new techniques. (1) In vitro culturing of (human) vascular endothelial cells has allowed studies on the subcellular localization for dimerization of pro-vWF subunits, multimerization, carbohydrate and proteolytic processing. Moreover, this approach has provided insight into the complex intracellular routing of vWF that proceeds by either one of two pathways. During the constitutive pathway, vWF is packaged in secretory vesicles that are rapidly secreted both at the luminal and at the basolateral side. The regulated pathway includes storage of vWF molecules in specialized organelles, i.e. the Weibel-Palade bodies. (2) The application of proteases to dissect purified multimeric vWF and to assign function(s) to defined proteolytic fragments of vWF. Monoclonal antibodies, raised against native vWF, that block specific functions and bind to fragments are subsequently employed to correlate structure and function. More precise localizations of functions are now feasible, using overlapping synthetic peptides derived from the primary amino acid sequence of (pro)-vWF and antibodies raised against such peptides. This approach has permitted a fine mapping of the interaction site of vWF with the platelet receptor glycoprotein (GP) IIB/IIIA. (3) Molecular cloning of full-length vWF cDNA and the development of eukaryotic expression systems have substantially increased the possibilities to investigate structures on pro-vWF involved in the biosynthesis and the assembly of multimers. In particular, the construction of point and deletion mutants of vWF, employing vWF cDNA, and subsequent expression in heterologous cells have demonstrated that proteolytic processing of pro-vWF, between arginine (Arg-763) and serine (Ser-764), to generate free propolypeptide and mature vWF is not required for multimerization. Finally, the propolypeptide has an obligatory role for the formation of multimers, enabling interdimer disulphide bonding of free sulphhydryl groups located within the mature vWF part of pro-vWF.

Biopolymers↗