Uncleaved Molecules Research Articles

Increased, Durable B-Cell and ADCC Responses Associated with T-Helper Cell Responses to HIV-1 Envelope in Macaques Vaccinated with gp140 Occluded at the CD4 Receptor Binding Site.

ABSTRACTStrategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens (Ag) for more long-lived, efficacious HIV-1 vaccine-induced B-cell responses. HIV-1 Env gp140 (native or uncleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells results in anergic effects on T-cell recruitment and consequently a lack of strong, robust, and durable B-memory responses. To test this hypothesis, we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3-kDa CD4 miniprotein mimetic, serving to block ligation of gp140 on CD4+ T cells while preserving CD4-inducible (CDi) neutralizing epitopes targeted by antibody-dependent cellular cytotoxicity (ADCC) effector responses. Importantly, immunization of rhesus macaques consistently gave superior B-cell (P < 0.001) response kinetics and superior ADCC (P < 0.014) in a group receiving the CD4bs-occluded vaccine compared to those of animals immunized with gp140. Of the cytokines examined, Ag-specific interleukin-4 (IL-4) T-helper enzyme-linked immunosorbent spot (ELISpot) assays of the CD4bs-occluded group increased earlier (P = 0.025) during the inductive phase. Importantly, CD4bs-occluded gp140 antigen induced superior B-cell and ADCC responses, and the elevated B-cell responses proved to be remarkably durable, lasting more than 60 weeks postimmunization.IMPORTANCE Attempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have been unsuccessful until now. Antigen-specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T cells as soluble shed antigen or as antigen-antibody complexes, causing impairment in the activation of these specialized CD4-positive T cells. We proposed that CD4-binding impairment is partly responsible for the relatively poor B-cell responses to HIV envelope-based vaccines. To test this hypothesis, we blocked the CD4 binding site of the envelope antigen and compared it to currently used unblocked envelope protein. We found superior and durable B-cell responses in macaques vaccinated with an occluded CD4 binding site on the HIV envelope antigen, demonstrating a potentially important new direction in future design of new HIV vaccines.

In vitro testing of a protease-sensitive contrast agent for optoacoustic imaging

We have designed a protease-sensitive imaging probe for optoacoustic imaging whose absorption spectrum changes upon cleavage by a protease of interest. The probe comprises an active site, a derivative of chlorophyll or natural photosynthetic bacteriochlorophyll that absorbs in the near infrared, conjugated to a peptide backbone specific to the protease being imaged. The uncleaved molecules tend to aggregate in dimers and trimers, causing a change in the absorption spectrum relative to that of the monomer. Upon cleavage, the probe molecules deaggregate, giving rise to a spectrum characteristic of monomers. We show using photospectrometry that the two forms of the probe have markedly different absorption spectra, which could allow for in vivo optoacoustic identification using a multiwavelength imaging strategy. Optoacoustic measurements using a narrow-band dye laser find spectral peaks in the two forms of the probe at the expected location. The optoacoustic signal from the uncleaved probe is found to be considerably weaker than that of the cleaved probe, perhaps due to poor optical-acoustic coupling in the aggregated molecules. However, ultimately, it is detection of the cleaved probe that is of the greatest import, since it reports on the protease activity of interest.

Site-specific N-glycosylation regulates the GPS auto-proteolysis of CD97

Auto-proteolysis at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is a hallmark of adhesion-GPCRs. Although defects in GPS auto-proteolysis have been linked to genetic disorders, information on its regulation remains elusive. Here, we investigated the GPS proteolysis of CD97, a human leukocyte-restricted and tumor-associated adhesion-GPCR. We found that CD97 is incompletely processed, unlike its close homolog, epidermal growth factor-like module-containing mucin-like hormone receptor 2. A unique pattern of N-glycosylation within the GPS motif of related adhesion-GPCRs was identified. The use of N-glycosylation inhibitors and mutants confirm site-specific N-glycosylation is an important determinant of GPS proteolysis in CD97. Our results suggest that N-glycosylation may regulate the processing of adhesion-GPCRs leading to the production of either cleaved or uncleaved molecules.

Vitellogenin of the parasitoid wasp, Encarsia formosa (Hymenoptera: Aphelinidae): gene organization and differential use by members of the genus

The vitellogenin (Vg) gene of the parasitoid wasp, Encarsia formosa (Hymenoptera: Aphelinidae), has been cloned and sequenced. The gene codes for a protein consisting of 1814 amino acids in seven exons. The position of the six introns in the E. formosa gene align with those inferred for the Vg gene of the honeybee, Apis mellifera. The position of two introns in the hymenopteran sequences are shared with every full-length insect Vg gene characterized to date. The deduced amino acid sequence of the E. formosa Vg gene most closely resembles that of the ichneumonid parasitoid, Pimpla nipponica (38% identity). The gene product, less the putative signal peptide, contains large quantities of serine (11.3% of total residues) but lacks the extensive polyserine tracts found in the Vgs of insects outside the apocritan Hymenoptera. The gene also codes for the highest level of lysine (9.5%), and lowest levels of phenylalanine (2.6%) and tyrosine (2.3%), observed in any insect Vg characterized to date. The mature gene product retains 12 cysteine residues in positions conserved in other insect Vgs. Ovary homogenates suggest that processed Vg is stored in the egg as an uncleaved molecule of approximately 200 kDa. Vg expression was examined in three additional Encarsia species. The protein was found in female E. sophia and E. luteola, but not in male E. luteola or female E. pergandiella. Despite extensive screening of a phage library prepared from E. pergandiella genomic DNA, a Vg gene was not detected in this species.

Mechanism of antibody-mediated reduction of nasopharyngeal colonization by Haemophilus influenzae type b studied in an infant rat model.

The mechanism of antibody-mediated reduction of Haemophilus influenzae type b (Hib) carriage was studied in the infant rat colonization model. Monoclonal Hib polysaccharide (PS) antibody (MAb) given intranasally or intraperitoneally and human secretory anti-Hib PS IgA given intranasally inhibited colonization by Hib during the entire follow-up period (2-48 h after challenge) but did not affect colonization by Hi, a noncapsulated variant of Hib. F(ab')2 fragments, prepared from the MAb or from human serum anti-Hib IgG reduced Hib colonization as efficiently as the uncleaved molecules. Complement depletion by cobra venom treatment had no effect on the antibody-mediated reduction of Hib colonization. These results indicate that Fc-mediated activities of immunoglobulins are not essential in the reduction of Hib colonization. Instead, antibodies to Hib most likely reduce colonization by a direct effect on growth of the bacteria or their adherence to the nasopharyngeal mucosa.

Cell surface expression of receptor protein tyrosine phosphatase RPTP mu is regulated by cell-cell contact.

RPTP mu is a transmembrane protein tyrosine phosphatase with an adhesion molecule-like ectodomain. It has recently been shown that RPTP mu mediates homophilic interactions when expressed in insect cells. In this study, we have examined how RPTP mu may function as a cell contact receptor in mink lung epithelial cells, which express RPTPmu endogenously, as well as in transfected 3T3 cells. We find that RPTP mu has a relatively short half-life (3-4 hours) and undergoes posttranslational cleavage into two noncovalently associated subunits, with both cleaved and uncleaved molecules being present on the cell surface (roughly at a 1:1 ratio); shedding of the ectodomain subunit is observed in exponentially growing cells. Immunofluorescence analysis reveals that surface expression of RPTPmu is restricted to regions of tight cell-cell contact. RPTPmu surface expression increases significantly with increasing cell density. This density-induced upregulation of RPTP mu is independent of its catalytic activity and is also observed when transcription is driven by a constitutive promoter, indicating that modulation of RPTPmu surface expression occurs posttranscriptionally. Based on our results, we propose the following model of RPTP mu function: In the absence of cell-cell contact, newly synthesized RPTP mu molecules are rapidly cleared from the cell surface. Cell-cell contact causes RPTPmu to be trapped at the surface through homophilic binding, resulting in accumulation of RPTP mu at intercellular contact regions. This contact-induced clustering of RPTPmu may then lead to tyrosine dephosphorylation of intracellular substrates at cell-cell contacts.

Interhelix geometry of stems I and II of a self-cleaving hammerhead RNA.

In order to investigate the geometry of a self-cleaving hammerhead domain, an RNA heteroduplex has been constructed in which two of the three helix stems of the domain have each been elongated to 76 duplex base pairs (bp), resulting in an RNA molecule of ca. 160 bp. The heteroduplex molecule is capable of undergoing self-cleavage at neutral pH, upon addition of either Mg2+ or Mn2+, but does not dissociate following cleavage. Using a combination of electrophoretic and hydrodynamic methods, as employed earlier to define the geometry of a four-way DNA branch [Copper & Hagerman (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7336-7340], we have determined that the elongated hammerhead stems are nearly collinear prior to self-cleavage. Following self-cleavage, and in the absence of Mg2+, the angle between the two stems becomes much more acute and/or flexible; however, in the presence of Mg2+, the cleaved structure appears to retain the geometry of the precleaved form (at least with respect to the interstem angle). It is also observed that the self-cleavage reaction is promoted by Mn2+ to a much greater extent than by Mg2+, consistent with earlier observations. Finally, although the elongated helices appear to be nearly collinear in the uncleaved molecule, the electrophoretic mobility of that species is dramatically reduced with respect to linear control RNAs, indicating that caution should be exercised in the quantitative assignment of branch angles solely from gel retardation experiments.

Purification and Characterization of a Strong Fibrinolytic Enzyme (Nattokinase) in the Vegetable Cheese Natto, a Popular Soybean Fermented Food in Japan

A strong fibrinolytic enzyme (nattokinase) was purified from the vegetable cheese natto. Nattokinase was extracted from natto with saline and isolated by sequential use of hydrophobic chromatography on Butyl-Toyopearl, ion-exchange chromatography on CM-Toyopearl, and gel-filtration on Sephadex G-50. The isolated protein gave a single sharp band on SDS-PAGE either before or after reduction. The sequence, as determined by automated Edman degradation of the uncleaved molecule and its enzymatically derived peptide, consisted of a total 275 amino acid residues (M.W=27,728) and exhibited a high homology with the subtilisins. The purified nattokinase digested not only fibrin but also several synthtic substrates. Among the synthetic substrates, the most sensitive substrate was Suc-Ala-Ala-Pro-Phe-pNA for subtilisin. PMSF inhibited both the fibrinolytic activity and the amidolytic activity. The results indicate that nattokinase is a subtilisin-like serine protease.

Complete amino acid sequence of streptococcal PepM49 protein, a nephritis-associated serotype. Conserved conformational design among sequentially distinct M protein serotypes.

The complete amino acid sequence of PepM49, a peptic fragment of the group A streptococcal type 49 M protein, the antiphagocytic cell surface molecule of the bacteria, is described. This fragment retains the opsonic antibody epitope of the native molecule. The sequence of PepM49, as determined by automated Edman degradations of the uncleaved molecule, and its tryptic and chymotryptic peptides, consists of a total of 143 residues (Mr = 17,187). PepM49, a nephritis-associated M protein serotype, exhibits significant internal homology in its sequence. However, identical sequence repeats of the kind seen in the rheumatic fever-associated serotypes M5, M6, and M24, are absent in PepM49. PepM49 exhibits varying degrees of homology with the M5, M6, and M24 proteins, which is consistent with the existence of variable and conserved regions in the M protein molecule. Predictive analysis as well as CD measurements revealed a high propensity of the PepM49 molecule to assume an alpha-helical conformation. Furthermore, a heptad periodicity of the nonpolar residues, a characteristic of alpha-helical coiled-coil proteins, extends over the entire length of the PepM49 protein. The differences in the nonpolar residue distribution divide the PepM49 sequence into three distinct domains, similar to those seen earlier in the M5 and M6 proteins. Together, these studies establish a conserved conformational design for the sequentially diverse M protein serotypes. However, the pattern of heptad periodicity in the PepM49 protein is quite distinct from that present in the PepM5 and M6 proteins, suggesting distinct differences in structural features among conformationally similar M protein serotypes. This may have relevance to the pathological differences associated with these M protein serotypes.

Thermal stability of human-fibroblast-collagenase-cleavage products of type-I and type-III collagens.

Rat skin type-I and type-III collagens were degraded by human fibroblast collagenase at a temperature below the 'melting' temperature for the two resulting fragments, namely the N-terminal three-fourths, TCA, and the C-terminal one-fourth, TCB. The specific cleavage of the collagen was confirmed by electrophoresis and determination of molecular length by electron microscopy. The two fragments were separated by gel filtration and the thermal stabilities of the isolated fragments were determined. For type-I collagen, the 'melting' temperatures of the two fragments were found to differ by only 0.5 degrees C and were 4.5-5.0 degrees C below that of the uncleaved molecule. The 'melting' temperatures of the uncleaved molecule and the N-terminal fragment were independent of the extent of N-terminal intramolecular cross-linking. For type-III collagen, the 'melting' temperatures of the fragments were found to differ by 1.3 degrees C. The small fragments of the two types of collagen 'melted' at the same temperature, whereas the large type-III fragment 'melted' at a slightly higher temperature than did the large type-I fragment. Reduction of the disulphide bonds located in the C-terminal type-III fragment did not affect the thermal stability of this fragment. The thermal stability of uncleaved type-III collagen was found to be variable, but the reason for this is not known at present.

Granular and extra-granular forms of immunoreactive calcitonin in normal rat "C" cells.

The intracellular localization of the intermediate calcitonin-related peptides produced during calcitonin maturation (CT, 3.4 Kd) from the polyprotein precursor (15 Kd) has been studied by means of subcellular fractionation of normal adult rat thyroid using a continuous hypertonic Percoll gradient. A fraction B (Bottom), containing the secretory granules, was separated from a fraction T (Top) constituted of rough and smooth membranes devoid of granules. Immunoreactive calcitonin extracted from both fractions was heterogeneous when analysed by gel electrophoresis followed by radioimmunoassay. In fraction T, the immunoreactive molecules assayed were associated with the membranes. Their apparent molecular weights (lower than 18 Kd) were compatible with the intermediates predicted by the aminoacid sequence of the precursor. However the maturation was not complete, as indicated by the absence of the monomer. The smallest molecular weight detected (4-4.5 Kd) was compatible with a calcitonin-katacalcin uncleaved molecule. These data strongly suggest the occurrence of a cleavage separating the N-terminal part of the molecule during the pre-granular step of calcitonin secretory pathway. In the granular fraction, the monomer constituted the granular matrix, representing 65% of the total immunoreactivity. The remainder was associated with the granules membranes, consisting of the 4-4.5 Kd precursor and some 43 Kd material which has already been demonstrated by others to be covalent polymers of calcitonin [17]. It is thus concluded that a second cleavage could take place in the granules where the major immunoreactive form is the processed calcitonin, either as free monomer or as covalent polymers associated with the membrane of the granules.

The complete amino acid sequence of a biologically active 197-residue fragment of M protein isolated from type 5 group A streptococci.

The complete amino acid sequence of a peptic fragment (Pep M5) of the group A streptococcal type 5 M protein, the antiphagocytic cell surface molecule of the bacteria, is described. This fragment, comprising nearly half of the native M molecule, is biologically active in that it has the ability to interact with opsonic antibodies as well as to evoke such an antibody response in rabbits. The sequence of Pep M5 was determined by automated Edman degradations of the uncleaved molecule and its enzymatically derived peptides. The primary peptides for Edman degradation were the arginine peptides obtained by tryptic digestion. The tryptic cleavage of Pep M5 was limited to the arginyl peptide bonds by derivatizing the epsilon-amino groups of lysine residues by reductive dihydroxypropylation. The overlapping peptides were generated by digestion of the unmodified Pep M5 with chymotrypsin, V8 protease, and subtilisin. The sequence thus established for the Pep M5 molecule consists of a total of 197 residues (Mr = 22,705). The Pep M5 protein contains some identical, or nearly so, repeating sequences: four 7-residue segments and two 10-residue segments. However, extensive sequence repeats of the kind previously reported within the partial sequence of another M protein serotype, namely Pep M24, were absent. The Pep M5 sequence is distinct from, but exhibits some homology with, the partial sequences of two other M protein serotypes, namely, Pep M6 and Pep M24. Furthermore, the 7-residue periodicity of the nonpolar and charged residues, an alpha-helical coiled-coil structural characteristic that was previously observed within the partial sequences of M proteins, was found to extend over a significant part of the Pep M5 sequence. The implication of these results to the function and immunological diversity in M proteins is discussed.

Separation of limited tryptic fragments of human ceruloplasmin by gel-permeation high-performance liquid chromatography

Limited tryptic proteolysis of human ceruloplasmin rapidly produces several large, protease-resistant fragments, suggesting that the molecule consists of several domains. In order to locate the sites of proteolytic cleavage in the whole molecule, we used gel-permeation high-performance liquid chromatography to determine the optimum conditions for fragment separation. Using a buffer containing 8 M urea, the 67,000-daltons tryptic fragment from single-chain ceruloplasmin was isolated in a sufficiently pure state for amino acid sequence analysis to determine its location in the uncleaved molecule. These results have been used in conjunction with amino acid sequence data to develop a schematic model of the domain structure of human ceruloplasmin.

Effect of monensin on Mason-Pfizer monkey virus glycoprotein synthesis.

The effect of the monovalent carboxylic ionophore monensin on the biosynthesis, intracellular transport, and surface expression of the glycoproteins of Mason-Pfizer monkey virus was examined. Cells treated with monensin at concentrations of 10(-7) or 10(-6) M continued to synthesize virus particles, which from electron microscopic studies appeared to bud normally from the plasma membrane of the cells. However, the particles released had an altered buoyant density in sucrose gradients and were noninfectious. These noninfectious virions had a normal complement of non-glycosylated polypeptides but showed a significantly reduced amount of glycosylated proteins. The gp70 and gp20 polypeptides appeared to be completely absent, and a heterogeneous, higher-molecular-weight protein was observed on the virions instead. Studies on intracellular protein synthesis indicated that the precursor (Pr86env) to gp70 and gp20 is synthesized normally but is not cleaved to the mature proteins. Immunofluorescence studies showed, however, that the uncleaved molecule is expressed on the cell surface. In this system, therefore, Mason-Pfizer monkey virus glycoprotein migration appears to occur in the presence of monensin, whereas the cleavage and insertion of the glycoproteins into virions are inhibited.