Loss Of Epitopes Research Articles

Immunohistochemical staging of neurofibrillary degeneration in Alzheimer's disease.

Antibodies to different phosphorylated and non-phosphorylated tau epitopes have been used to identify three histologically distinct types of neurofibrillary tangles in Alzheimer's disease. Intracellular tangles (Type 1) were identified by antibodies recognizing epitopes throughout the tau molecule, including the NH2-terminus. Compact extracellular tangles (Type 2) were characterized by the loss of NH2-terminal immunoreactivity and retention of other tau epitopes. Dispersed extracellular tangles (Type 3) were characterized by the presence of epitopes associated with the microtubule binding region and the COOH-terminus. These three types of tangles, found in situ in hippocampus, could be created experimentally by proteolytic treatment of brain sections. These findings suggest that three stages of neurofibrillary degeneration can be understood as a sequential stripping of paired helical filaments in which the loss of amino-terminus epitopes, followed by loss of phosphorylated epitopes, results in the appearance of dispersed extracellular tangles containing PHF-core epitopes.

Analysis of toxinogenic functions associated with the RTX repeat region and monoclonal antibody D12 epitope of Escherichia coli hemolysin.

Amino acids (aa) 550 through 850 of the Escherichia coli hemolysin (HlyA) contain sequences important for several steps in cytolysis. These include the Ca(2+)-binding glycine-rich tandem repeats recognized by the monoclonal antibody A10, the putative HlyC-dependent acylation site that corresponds to the monoclonal antibody D12 epitope, and the erythrocyte specificity domain which confers erythrolytic activity to the Pasteurella haemolytica leukotoxin. To further investigate the toxinogenic functions associated with this region of HlyA, we constructed mutants in the hlyA sequences coding for the repeat region and the D12 epitope. Mutants were analyzed for anti-HlyA antibody reactivity, cytolytic activities, target cell binding, Ca2+ requirements, and virulence. The D12 epitope was mapped to aa 673 through 726, with portions of the epitope both amino terminal and carboxy terminal to aa 700. This region was necessary, but not sufficient, for toxin binding to erythrocytes. A substitution at aa 684 resulted in loss of the D12 epitope, while cytolytic activity was retained. The nature of the D12 epitope and its associated functions are discussed. The A10 epitope mapped to residues 745 through 829, corresponding to repeats 4 through 11. Insertions within the glycine-rich repeats resulted in mutant forms of HlyA which retained A10 reactivity but required increased Ca2+ for lytic activity. These in vitro effects on cytolysis corresponded to a significant decrease in HlyA-mediated virulence in mice. HlyA from one insertion mutant was able to associate with leukocyte membranes under conditions that were Ca2+ deficient for cytolysis. The role of the glycine-rich repeats and Ca2+ in HlyA activity are discussed.

Loss of neurofilament epitopes from axons of Lewy body-containing neurons : M.M. O'Neill and W.D. Hill. Dept. of Cell. Biol. and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA

Loss of neurofilament epitopes from axons of Lewy body-containing neurons : M.M. O'Neill and W.D. Hill. Dept. of Cell. Biol. and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA

Decreased Opsin mRNA and Immunoreactivity in Progressive Rod-Cone Degeneration (prcd): Cytochemical Studies of Early Disease and Degeneration

Opsin mRNA level and immunoreactivity were examined by in situ hybridization and immunocytochemistry in normal and progressive rod cone degeneration (prcd)-affected dogs. In situ hybridization used 35S- and/or 3 H-labeled bovine opsin cRNA probes; immunocytochemistry used six monoclonal mouse anti-bovine opsin antibodies (MAb1) that are specific to different regions of the N-terminal, loop v-vi and the C-terminal domains. Optimal labeling and histological resolution at the single cell level were achieved with semi-thin sections of DGD wax-embedded tissues; it was possible to correlate the cytochemical observations with the disease staging in topographically defined regions that exhibited different disease severity. In early disease (stages 0-1), opsin mRNA levels and immunoreactivity were normal. During the transition from disease to degeneration (stage 2), however, opsin mRNA was reduced sharply; it then rapidly became undetectable in late stages of degeneration (stages 3, 4). Reduction of immunoreactivity was seen with all the MAbs in stages 2 and 3, but the degree of reduction varied remarkably in different regions of the protein molecule; immunoreactivity was reduced more in the cytoplasmic regions, particularly in the phosphorylation sites and the far end of the C-terminal domain. In contrast, the epitopes of the N-terminal domain that are located in the intradiscal compartment were better preserved. It is noteworthy that, in stages 2 and 3, many rod cells still survived despite the decrease in the mRNA level and immunoreactivity. The results indicate that early disease in prcd-affected rods is not initiated by a reduction of opsin mRNA or the protein quantity. However, opsin expression disappears in early degeneration and before cell death. The differences in immunoreactivity with disease may result either from alterations in the protein structure or configuration, or from selective loss of epitopes located in the cytoplasmic domains of the molecule.

Loss of a neutralizing epitope by a spontaneous point mutation in the V3 loop of HIV-1 isolated from an infected laboratory worker.

The third hypervariable region, or V3 loop, represents the principal neutralizing domain of the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequential viral isolates from a laboratory worker (LW) accidentally infected with HIV-1IIIB in 1985 were analyzed using type-specific neutralizing monoclonal antibodies directed to the V3 loop. A single amino acid substitution, Ala-->Thr at position 21 in the V3 loop of HIV-1LW isolated in 1987, was shown to determine the loss of the neutralizing epitope recognized by one of the monoclonal antibodies (M77). However, this antibody efficiently recognized linear V3 loop peptides containing either the Ala or Thr residue at position 21, indicating that a local change in conformation was responsible for the epitope loss in the native gp120. Molecular modeling studies, experimentally supported by different amino acid replacements at position 21, indicated that the Ala-->Thr substitution leads to a drastic change in the domain of the V3 loop, which contains the complementary surface for antibody binding. These results provide evidence for the first time that a conformation-dependent epitope within the V3 loop of HIV-1 is involved in the generation of neutralization escape mutants in vivo.

Structure, function, and regulation of the enzyme activity of prostate-specific antigen.

Prostate-specific antigen (PSA) and human glandular kallikrein 1 (hGK-1) are structurally similar products of the human glandular kallikrein gene locus on chromosome 19 that become selectively expressed by human prostate tissue. PSA is one of the most abundant prostate-derived proteins in the seminal fluid. The mature form of PSA, a single-chain glycoprotein of 237 amino acids, is a serine protease manifesting restricted chymotrypsin-like activity. PSA is mainly responsible for gel dissolution in freshly ejaculated semen by proteolysis of the major gel-forming proteins semenogelin I and II and fibronectin. PSA complexed to alpha 1-antichymotrypsin (ACT) is the predominant molecular form of serum PSA, although complex formation is slow between the purified proteins in vitro. PSA also forms stable complexes with alpha 2-macroglobulin (alpha 2M) in vitro, but as this results in encapsulation of PSA and complete loss of the PSA epitopes, the in vivo significance of this complex formation is presently unclear. A free, noncomplexed form of PSA constitutes a minor fraction of the serum PSA, although serum ACT occurs at large molar excess.

Rotavirus vaccine administered parenterally induces protective immunity.

We performed experiments to determine whether parenteral immunization with SA11 rotavirus can induce active protective immunity in a rabbit model of rotavirus infection. After one or two intramuscular injections of 1 ml of live or formalin-inactivated SA11 virus, we evaluated the mucosal and serologic immune response and protection from challenge with a high dose of live, virulent rabbit (Ala) rotavirus. Inactivated SA11 virus preparations, evaluated by enzyme-linked immunosorbent assay (ELISA) with a panel of VP4- and VP7-specific neutralizing and nonneutralizing monoclonal antibodies, did not show a loss of epitopes from the inactivation procedure compared with live virus. Administration of two doses of vaccine, one at zero days postvaccination (DPV) and a booster shot at 49 DPV, followed by challenge at 71 DPV with 3.5 x 10(5) PFU of Ala virus resulted in protection from challenge. None of the two-dose virus-vaccinated rabbits shed virus after challenge, while virus shedding was detected in all control rabbits (P = 0.001, Fisher's exact two-tailed test). Differences in total serum immunoglobulin (Ig) antirotavirus ELISA titers (P < 0.05, Wilcoxon's rank sum test) were observed between groups vaccinated with virus in aluminum phosphate or Freund's adjuvant but not between groups vaccinated with live or inactivated virus in either adjuvant. All rabbits given two doses of vaccine had detectable antirotavirus intestinal antibody of the IgG, but not IgA, isotype. After challenge, fourfold or greater increases in intestinal IgG antibody responses were observed in three rabbits, whereas all controls and all but one virus-vaccinated rabbit had an intestinal IgA antibody response. In contrast, vaccination of rabbits with one dose of SA11 followed by challenge at 21 DPV did not protect from challenge; no difference in the mean number of days of virus shedding between any of the vaccinated groups and controls was observed. A serologic, but not a mucosal, antibody response was observed after the one-dose vaccination regimen. Differences in serologic antibody titers were not observed between any of the one-dose virus-vaccinated groups. These data indicate that parenteral vaccination with two, but not one, doses of rotavirus in either Freund's adjuvant or aluminum phosphate can induce active protection from challenge.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkali hydrolysis of recombinant proteins allows for the rapid identification of class I MHC-restricted CTL epitopes.

The characterization of the epitopes recognized by CTL provides insights into the nature of protective immune responses and facilitates the development of methods to enhance immunity to human pathogens. However, no easily applicable approach for CTL epitope identification has been developed. We present a rapid and efficient method for locating CTL epitopes within a protein. The gene encoding the protein of interest is inserted into an inducible prokaryotic expression vector. Random peptides are then generated by alkali digestion of intact or lysed Escherichia coli expressing the protein and assayed for the presence of the epitope by coating target cells for a standard CTL targeting assay. A large panel of clones containing serial 3'-deletions of the gene is then generated by exonuclease III digestion, and the expressed truncated proteins are similarly analyzed for the presence of the antigenic peptide. The epitope is located by determining the deletion points of clones expressing sequential truncations and differing in Ag expression. This technique was used to identify the H-2Ld-restricted nonamer in E. coli beta-galactosidase, with residues 876-884 representing the naturally processed epitope. To test the applicability of this method to other proteins, two genes from human CMV, an often fatal pathogen in immunocompromised individuals, were screened for HLA class I-restricted epitopes. An HLA-B18-restricted epitope from the CMV major immediate-early protein was found to lie between residues 378 and 389, and an HLA-B35-restricted epitope from the CMV pp65 matrix protein was characterized as residues 123 to 131. The results demonstrate that this technique can be used to rapidly identify CTL epitopes within a chosen protein and should be useful for assaying viral isolates or neoplasms for loss of epitopes after mutation and selection by host immune responses.

Dynamics of peptide binding to purified antibody-bound H-2Db and H-2Db beta 2m complexes.

Although it is clear that each component of the class I MHC trimolecular complex (heavy chain, beta 2m, and antigenic peptide) contributes to its formation and stability, the specific interaction governing assembly and disassembly remain to be clarified. In an effort to address these issues using purified H-2Db molecules, we used a solid-phase binding assay recently developed in our laboratory to quantify kinetic parameters for class I assembly and disassembly. It was found that the influenza NP peptide Y367-374 associated with preformed empty complexes of 28-14-8S- (i.e., anti-alpha 3) bound Db beta 2m dimers much more quickly (t 1/2 < 0.2 h at 22 degrees C) than it did when coincubated with an anti-alpha 3 bound Db and human beta 2m (t1/2 3.5 h at 22 degrees C). The previously reported potential for the NP peptide Y367-374 to interact directly with free Db heavy chains and configure the conventionally beta 2m-dependent B22 epitope in the absence of beta 2m, was confirmed using our assay system. However, the rate of B22 epitope formation induced in the Db heavy chain by NP Y367-374 was considerably slower (t1/2 13 h, at 22 degrees C) and much less efficient on a molar basis than that resulting from the addition of beta2m (t1/2, 0.75 h, at 22 degrees C). In contrast, the Db heavy chain with NP-Y367-374 was more resistant to thermal disassembly (as measured by loss of the B22 epitope, t1/2 2h, 37 degrees C) than the Db beta 2m empty dimer (t1/2 0.2 h). Finally, stability of the preformed trimolecular complex of heavy chain, beta 2m, and peptide was found to diminish in accordance with deviation of the peptide from the optimal length and with increasing temperature from 4 to 37 degrees C.

Bile acid transport into hepatocyte smooth endoplasmic reticulum vesicles is mediated by microsomal epoxide hydrolase, a membrane protein exhibiting two distinct topological orientations.

Bile acids, such as taurocholate, have been shown to be transported into hepatocyte smooth endoplasmic reticulum (SER) vesicles. This process is Na(+)-independent, electrogenic, inhibitable by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and taurochenodeoxycholate, with a Km of 352 microM and a Vmax of 29.6 nmol/mg protein/min. The observed transport is mediated by the bifunctional protein, microsomal epoxide hydrolase (mEH) which can also mediate bile acid transport into hepatocytes across the sinusoidal plasma membrane (von Dippe, P., Amoui, M., Alves, C., and Levy, D. (1993) Am. J. Physiol. 264, G528-G534). mEH was isolated from SER membranes by immunoprecipitation with monoclonal antibody (mAb) 25D-1 which recognizes this protein on the surface of intact hepatocytes. The SER-derived protein exhibited an apparent molecular weight, isoelectric point, N-terminal amino acid sequence, and mEH-specific activity that were indistinguishable from the plasma membrane form of the enzyme. Proteoliposome reconstitution of the SER taurocholate transport system indicated that mEH was absolutely required for the expression of transport capacity. The interaction of mAb 25D-1 with mEH on intact right-side-out SER vesicles demonstrated that the epitope found on the surface of hepatocytes was also found on the cytoplasmic surface of these vesicles (80%) and in the lumen (20%) suggesting the presence of two forms of this protein in the SER, the latter from being sorted to the cell surface. The existence of two orientations of this protein in the SER was confirmed by the sensitivity to tryptic digestion, where 75% of the mAb epitope was accessible to the enzyme. The loss of the 25D-1 epitope correlated with loss of taurocholate transport capacity. The role of mEH in the transport process and the orientation of the transporting isoform was further established by demonstrating that mAb 25A-3, which also reacts with mEH on the hepatocyte surface, was able to directly inhibit taurocholate transport in the SER vesicle system. These and previous results thus establish that isoforms of mEH can mediate taurocholate transport at the sinusoidal plasma membrane and in SER vesicles and that this bifunctional protein can exist in two orientations in the SER membrane. The association of bile acids with the SER suggests a possible role of intracellular vesicles in the transhepatocellular movement of bile acids from the sinusoidal to the canalicular compartment.

Congenital nephrosis of the Finnish type (CNF): matrix components of the glomerular basement membranes and of cultured mesangial cells.

Congenital nephrosis of the Finnish type (CNF) is a hereditary renal disease of unknown aetiology manifested by massive proteinuria of the newborn and unresponsive to any treatment. In this study kidney samples and cultured glomerular mesangial cells from five patients with CNF were studied by indirect immunofluorescence microscopy for the presence and location of major basement membrane matrix (GBM) components. Histological changes of glomeruli ranging from mild thickening of basement membranes to total obliteration and sclerosis were seen. Notably, thickening of the subepithelial layer of Bowman's capsules was regularly seen along with hypercellularity at the juxtaglomerular areas. The matrix components studied (laminin, plasma- and cellular fibronectin, type IV collagen, including the NC-1, alpha-1 and alpha-3 chains, heparan sulphate proteoglycan (HSPG) core protein, thrombospondin) were characteristically seen within the glomeruli. Local thickenings alternating with total loss of epitopes along the GBM were seen, especially with anti-type IV collagen and anti-HSPG antibodies. Sera from CNF patients after transplantation failed to show antibodies against GBM structures in immunofluorescence microscopy, suggesting that no missing epitopes of GBM are introduced with the transplant kidney. Cultured mesangial cells of CNF glomeruli also showed continued in vitro production of the matrix components and their incorporation into the matrix underneath the cell layer.

Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme.

Monoclonal antibodies (mAbs) have been used to study structure-function relationships of (R)-3-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a lipid-requiring mitochondrial membrane enzyme with an absolute and specific requirement for phosphatidylcholine (PC) for enzymic activity. The purified enzyme (apoBDH, devoid of phospholipid and thereby inactive) can be re-activated with preformed phospholipid vesicles containing PC or by short-chain soluble PC. Five of six mAbs cross-react with BDH from bovine heart and rat liver, including two mAbs to conformational epitopes. One mAb was found to be specific for the C-terminal sequence of BDH and served to: (1) map endopeptidase cleavage and epitope sites on BDH; and (2) demonstrate that the C-terminus is essential for the activity of BDH. Carboxypeptidase cleavage of only a few (< or = 14) C-terminal amino acids from apoBDH (as detected by the loss of C-terminal epitope for mAb 3-10A) prevents activation by either bilayer or soluble PC. Further, for BDH in bilayers containing PC, the C-terminus is protected from carboxy-peptidase cleavage, whereas in bilayers devoid of PC the C-terminus is cleaved, and subsequent activation by PC is precluded. We conclude that: (1) the C-terminus of BDH is essential for enzymic activity, consistent with the prediction, from primary sequence analysis, that the PC-binding site is in the C-terminal domain of BDH; and (2) the allosteric activation of BDH by PC in bilayers protects the C-terminus from carboxypeptidase cleavage, indicative of a PC-induced conformational change in the enzyme.

Dimerization of CD4-C κ chimeric molecules leads to loss of CD4 epitopes

Structural similarities between two members of the immunoglobulin superfamily were explored by making chimeric immunoglobulin/CD4 antigen molecules. A crossover in the middle of the originally proposed J κ homology unit of the first domain of the CD4 molecule was used to construct a chimeric molecule having human and mouse CD4 antigen sequence through the first 108 amino acids and murine J κ and C κ sequence thereafter. This molecule was expressed in the presence and absence of an immunoglobulin heavy chain. The resulting proteins were assayed for the expression of CD4 epitopes that should be present based on epitope mapping data. Monomeric, homodimeric, and heavy chain/light chain tetrameric forms of the recombinant protein were secreted and were all detectable with anti-kappa reagents. CD4 antibodies precipitated only the form of the CD4-C κ light chain protein which appears as a monomer by polyacrylamide gel electrophoresis. Neither the homodimer nor the heavy chain/light chain tetramer were detected with CD4 monoclonal antibodies. An engineered gene having this CD4 antigen first domain joined to the human IgGI constant region, when coexpressed with a mouse lambda light chain, also failed to express detectable CD4 epitopes. The structural implications of the presence or absence of CD4 epitopes on these proteins is discussed.

An Immunomagnetic Cell Separation Technique for Isolating Oligodendroglial Progenitor Cells of Trout CNS

An immunomagnetic cell separation technique for the rapid and efficient isolation of oligodendroglial precursor cells dissociated from the larval trout brain is described. Following preliminary enrichment by Percoll density gradient centrifugation, the dissociated cells are incubated with the monoclonal antibody A2B5 and biotinylated anti-mouse IgM and finally labeled with streptavidin-coated superparamagnetic beads. After passage through a magnetic field, a 94 to 98% pure population of A2B5+ cells is obtained with up to 89% recovery. When cultured, the cells rapidly regenerate their cytoplasmic processes and adopt a characteristic bipolar or tripolar morphology closely resembling that of oligodendroglial progenitors of the mammalian brain. During the first week in culture, many of the cells differentiate into oligodendrocytes, as reflected by the loss of the A2B5 epitope and expression of the early myelin marker 6D2. In an alternative approach, the monoclonal antibody 6D2 is used to effect a complete removal of differentiated oligodendrocytes from cell dissociates by negative immunoselection.

Specific regions of Escherichia coli OmpF protein involved in antigenic and colicin receptor sites and in stable trimerization

Four different mutations were obtained by selecting for resistance to colicin N and screening for continued production of the OmpF protein of Escherichia coli. Two of them also conferred resistance to colicin A. The substitutions C for R-168 (R168C) and E284K caused the loss of the E21 epitope, while the transition G285D altered the E18, E19, and E20 antigenic sites. The substitution G119D drastically affected the stability of the trimeric conformation.

A stromelysin assay for the assessment of metalloprotease inhibitors on human aggregated proteoglycan.

Human proteoglycan was aggregated to an immobilized hyaluronan solid phase on a 96-well ELISA plate. This complex was then degraded by recombinant human stromelysin. The remaining proteoglycan fragments were detected using a monoclonal antibody probe directed against the chondroitin sulfate (CS) region of the core protein. Stromelysin degraded the aggregate in a time and dose dependent manner as reflected by the loss of the CS epitope. Assay sensitivity was 0.125 U/well with total loss of the CS epitope occurring at 4 U/well. o-phenanthroline (IC50 = 52 microM) and U24522 (IC50 = 9 microM) inhibited degradation, while phosphoramidon did not. Serine and cysteine protease inhibitors had no effect. A comparative analysis of this assay with a reference method, substance P assay, gave similar inhibitor profiles. The use of aggregated human proteoglycan (native conformation) as a substrate, may better reflect how stromelysin inhibitors behave in the presence of complex substrates such as cartilage matrix.

Loss of Antigenic Epitopes as the Result of Env Gene Recombination in Retrovirus-Induced Leukemia in Immunocompetent Mice

The murine leukemia virus, E-55 + virus, induces a thymic lymphoma/leukemia in 100% of BALB.K mice infected as adults after a latent period of 4 months or more (Pozsgay et al., Virology 173, 330-334, 1989). Two molecular clones of virus designated E-55 + and E-55 - based on their ability to encode the E-55 epitope detected by the monoclonal antibody 55 (mAb 55) were isolated from a leukemic BALB.K mouse inoculated with a biologically cloned E-55 + virus (Chesebro et al., Virology 112, 131-144, 1981). Env gene sequence analysis of E-55 + and E-55 - clones showed that the E-55 - virus was generated from the E-55 + virus as the result of a recombination between E-55 + virus and the endogenous ecotropic virus, emv-1, carried in the genome of the BALB.K mouse strain. The recombinant E-55 - virus is replication competent. This recombination event and the consequential expression of E-55 - virus consistently occur in immunocompetent BALB.K mice inoculated with the E-55 + virus and appear to play a role in the loss of epitopes recognized by virus neutralizing antibodies. The loss of these epitopes apparently allows the virus to evade the host immune response.

Interaction between Aspergillus fumigatus and basement membrane laminin: Binding and substrate degradation

Aspergillus fumigatus, the causative agent of human aspergillosis, binds to and degrades basement membrane laminin. Using immunoelectron microscopy, laminin binding appeared to be associated with the cell wall expansions of resting conidia, and progressively extended to the outer electron dense layer of the conidial wall during the germination process. Labeling of thin sections revealed numerous binding sites in the cytoplasm, whereas the inner cell wall and the plasma membrane were not labeled. Attachment of A fumigatus conidia on microtiter plates coated with laminin and its fragments P1 and E8 was also investigated. Conidia cells showed good adhesion to wells coated with laminin. As indicated by inhibition experiments, the interaction was specific and fragment P1 represented the major binding site on the laminin molecule. In addition, since A fumigatus produced an extracellular serine protease, we determined the susceptibility of laminin to this enzyme. We demonstrated that protease extract was capable to degrade laminin in solution as well as in tissue sections. The laminin cleavage products were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All the three chains were extensively degraded within 1 h. Treatment of the crude protease extract with the enzyme inhibitors, phenylmethylsulfonyl-fluoride and chymostatin, blocked the degradation of laminin, indicating a chymotrypsin-like serine protease activity. Immunofluorescence microscopy of cryostat sections of mouse and rat kidneys treated with the protease extract showed widespread loss of laminin epitopes from basement membranes. Enzyme treatment also removed immunoreactivity from lungs as observed after immunoperoxidase performed on paraffin sections. Binding and proteolytic degradation of laminin may together facilitate initial interaction of A fumigatus with the host tissues.

5] Protein conformational changes in virus-cell fusion

Publisher Summary This chapter discusses the protein conformational changes in virus- cell fusion. With the techniques described in this chapter, it is possible to examine what happens to a relatively small region of the viral protein during its conformational change—that is, the loss of an antigenic epitope, or the exposure of a particular cleavage site or disulfide bond. Detailed kinetic studies, and perhaps the use of low temperatures, may reveal the sequence with which these rearrangements occur. In addition to describing aspects of the conformational change, it is necessary to ask whether the changes observed in an in vitro system are relevant for fusion in vitro. The conditions under which a given aspect of the conformational change occurs should reflect those under which fusion takes place. Most studies on the “acid” conformations of viral fusion proteins have examined structural changes that occur after acid treatment and reneutralization. The resulting structures may of course differ in important respects from the nascent molecules shortly after acid treatment.

Loss of laminin epitopes during glomerular basement membrane assembly in developing mouse kidneys.

Kidney glomerular basement membranes (GMBs) originate in development from fusion of a dual basement membrane between endothelial cells and primitive epithelial podocytes. After fusion, segments of newly synthesized matrix, derived primarily from podocytes, appear as subepithelial outpockets and are spliced into GBMs during glomerular capillary loop expansion. To investigate GBM assembly further, we examined newborn mouse kidneys with monoclonal rat anti-mouse laminin IgGs (MAb) conjugated to horseradish peroxidase (HRP). In adults, these MAb strongly label glomerular mesangial matrices but bind only weakly or not at all to mature GBMs. In contrast, anti-laminin MAb intensely bound newborn mouse GBMs undergoing initial assembly. After intraperitoneal injection of MAb-HRP into neonates, dense binding occurred across both subendothelial and subepithelial pre-fusion GMBs as well as forming mesangial matrices. Considerably less MAb binding was seen, however, in post-fusion GBMs from more mature glomeruli in the same section, although mesangial matrices remained positive. In addition, new subepithelial segments in areas of splicing were negative. These results conflict with those obtained previously with injections of polyclonal anti-laminin IgGs into newborns or adults, which result in complete labeling of all GBMs. Although epitope masking cannot be completely excluded, we believe that decreased MAb binding to developing GBM reflects actual epitope loss. This loss could occur by laminin isoform substitution, conformational change, and/or proteolytic processing during GBM assembly.