scholarly journals Investigating Constraints Along the Plant Secretory Pathway to Improve Production of a SARS-CoV-2 Spike Vaccine Candidate

2022 ◽  
Vol 12 ◽  
Author(s):  
Emmanuel Margolin ◽  
Matthew Verbeek ◽  
Warren de Moor ◽  
Ros Chapman ◽  
Ann Meyers ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Vaccine Development ◽  
Molecular Farming ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Efficient Production ◽  
Emerging Viruses ◽  
Viral Glycoprotein ◽  
Viral Glycoproteins ◽  
Efficient Processing

Given the complex maturation requirements of viral glycoproteins and the challenge they often pose for expression in plants, the identification of host constraints precluding their efficient production is a priority for the molecular farming of vaccines. Building on previous work to improve viral glycoprotein production in plants, we investigated the production of a soluble SARS-CoV-2 spike comprising the ectopic portion of the glycoprotein. This was successfully transiently expressed in N. benthamiana by co-expressing the human lectin-binding chaperone calreticulin, which substantially increased the accumulation of the glycoprotein. The spike was mostly unprocessed unless the protease furin was co-expressed which resulted in highly efficient processing of the glycoprotein. Co-expression of several broad-spectrum protease inhibitors did not improve accumulation of the protein any further. The protein was successfully purified by affinity chromatography and gel filtration, although the purified product was heterogenous and the yields were low. Immunogenicity of the antigen was tested in BALB/c mice, and cellular and antibody responses were elicited after low dose inoculation with the adjuvanted protein. This work constitutes an important proof-of-concept for host plant engineering in the context of rapid vaccine development for SARS-CoV-2 and other emerging viruses.

scholarly journals A Roadmap for the Molecular Farming of Viral Glycoprotein Vaccines: Engineering Glycosylation and Glycosylation-Directed Folding

2020 ◽  
Vol 11 ◽  
Author(s):  
Emmanuel Margolin ◽  
Max Crispin ◽  
Ann Meyers ◽  
Ros Chapman ◽  
Edward P. Rybicki
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Molecular Farming ◽  
Molecular Engineering ◽  
Early Years ◽  
In Planta ◽  
Post Translational Modification ◽  
Viral Glycoprotein ◽  
Viral Glycoproteins ◽  
Cellular Machinery

Immunization with recombinant glycoprotein-based vaccines is a promising approach to induce protective immunity against viruses. However, the complex biosynthetic maturation requirements of these glycoproteins typically necessitate their production in mammalian cells to support their folding and post-translational modification. Despite these clear advantages, the incumbent costs and infrastructure requirements with this approach can be prohibitive in developing countries, and the production scales and timelines may prove limiting when applying these production systems to the control of pandemic viral outbreaks. Plant molecular farming of viral glycoproteins has been suggested as a cheap and rapidly scalable alternative production system, with the potential to perform post-translational modifications that are comparable to mammalian cells. Consequently, plant-produced glycoprotein vaccines for seasonal and pandemic influenza have shown promise in clinical trials, and vaccine candidates against the newly emergent severe acute respiratory syndrome coronavirus-2 have entered into late stage preclinical and clinical testing. However, many other viral glycoproteins accumulate poorly in plants, and are not appropriately processed along the secretory pathway due to differences in the host cellular machinery. Furthermore, plant-derived glycoproteins often contain glycoforms that are antigenically distinct from those present on the native virus, and may also be under-glycosylated in some instances. Recent advances in the field have increased the complexity and yields of biologics that can be produced in plants, and have now enabled the expression of many viral glycoproteins which could not previously be produced in plant systems. In contrast to the empirical optimization that predominated during the early years of molecular farming, the next generation of plant-made products are being produced by developing rational, tailor-made approaches to support their production. This has involved the elimination of plant-specific glycoforms and the introduction into plants of elements of the biosynthetic machinery from different expression hosts. These approaches have resulted in the production of mammalian N-linked glycans and the formation of O-glycan moieties in planta. More recently, plant molecular engineering approaches have also been applied to improve the glycan occupancy of proteins which are not appropriately glycosylated, and to support the folding and processing of viral glycoproteins where the cellular machinery differs from the usual expression host of the protein. Here we highlight recent achievements and remaining challenges in glycoengineering and the engineering of glycosylation-directed folding pathways in plants, and discuss how these can be applied to produce recombinant viral glycoproteins vaccines.

scholarly journals Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

1984 ◽  
Vol 32 (7) ◽  
pp. 690-696 ◽  
Author(s):  
J Fischer ◽  
G Uhlenbruck ◽  
P J Klein ◽  
M Vierbuchen ◽  
R Fischer
Keyword(s):  
Molecular Weight ◽  
Gastric Mucosa ◽  
High Molecular Weight ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Gastric Cancer Cells ◽  
Tissue Sections ◽  
Gradient Gel Electrophoresis ◽  
Triton X

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

scholarly journals Retrograde Transport of Golgi-localized Proteins to the ER

1998 ◽  
Vol 140 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Nelson B. Cole ◽  
Jan Ellenberg ◽  
Jia Song ◽  
Diane DiEuliis ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Viral Glycoprotein ◽  
Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

scholarly journals Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
George Ueda ◽  
Aleksandar Antanasijevic ◽  
Jorge A Fallas ◽  
William Sheffler ◽  
Jeffrey Copps ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
De Novo ◽  
Viral Glycoprotein ◽  
Influenza Hemagglutinin ◽  
Self Assembling ◽  
Cryo Electron Microscopy ◽  
Viral Glycoproteins ◽  
Protein Nanoparticles ◽  
New Generation ◽  
Tailored Design

Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first de novo designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.

scholarly journals Stability of the HSV-2 US-6 Gene in the del II, del III, CP77, and I8R-G1L Sites in Modified Vaccinia Virus Ankara After Serial Passage of Recombinant Vectors in Cells

Vaccines ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 137 ◽  
Author(s):  
Vajini N. Atukorale ◽  
Jerry P. Weir ◽  
Clement A. Meseda
Keyword(s):  
Vaccinia Virus ◽  
Intergenic Region ◽  
Vaccine Development ◽  
Gc Content ◽  
Serial Passage ◽  
Sequencing Analysis ◽  
Viral Glycoprotein ◽  
Production Scale ◽  
Modified Vaccinia Virus Ankara ◽  
In Cells

The modified vaccinia virus Ankara (MVA), a severely attenuated strain of vaccinia virus, is a promising vector platform for viral-vectored vaccine development because of its attributes of efficient transgene expression and safety profile, among others. Thus, transgene stability in MVA is important to assure immunogenicity and efficacy. The global GC content of the MVA genome is 33%, and GC-rich sequences containing runs of C or G nucleotides have been reported to be less stable with passage of MVA vectors in cells. The production of recombinant MVA vaccines requires a number of expansion steps in cell culture, depending on production scale. We assessed the effect of extensive passage of four recombinant MVA vectors on the stability of the GC-rich herpes simplex type 2 (HSV-2) US6 gene encoding viral glycoprotein D (gD2) inserted at four different genomic sites, including the deletion (del) II and del III sites, the CP77 gene locus (MVA_009–MVA_013) and the I8R-G1L intergenic region. Our data indicate that after 35 passages, there was a reduction in gD2 expression from del II, del III and CP77 sites. Sequencing analysis implicated US6 deletion and mutational events as responsible for the loss of gD2 expression. By contrast, 85.9% of recombinant plaques expressed gD2 from the I8R-G1L site, suggesting better accommodation of transgenes in this intergenic region. Thus, the I8R-G1L intergenic region may be more useful for transgene insertion for enhanced stability.

scholarly journals Two classes of mutant mammary tumor virus-infected HTC cell with defects in glucocorticoid-regulated gene expression.

1983 ◽  
Vol 3 (2) ◽  
pp. 149-160 ◽  
Author(s):  
G L Firestone ◽  
K R Yamamoto
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Mammary Tumor ◽  
Hormonal Regulation ◽  
Wild Type ◽  
Viral Glycoprotein ◽  
Mammary Tumor Virus ◽  
Viral Glycoproteins ◽  
Tumor Virus ◽  
Regulated Gene Expression

We have isolated mutant derivatives of M1.54 (a mammary tumor virus [MTV]-infected rat hepatoma [HTC] cell line containing multiple integrated proviruses) that fail to express hormone-inducible cell surface viral glycoproteins. In wild-type M1.54, the synthetic glucocorticoid dexamethasone selectively stimulates the rate of synthesis of MTV RNA. In addition, dexamethasone is essential for posttranslational maturation of three of the four cell surface viral glycoproteins processed from the MTV glycosylated precursor polyprotein; the fourth mature species is produced constitutively. Two mutant phenotypes are described; each contains glucocorticoid receptors that are indistinguishable from the wild-type receptor with respect to hormone affinity, intracellular concentration, nuclear translocation efficiency, DNA-cellulose chromatography, and sedimentation rate. In one class, represented by the mutant line CR1, dexamethasone fails to stimulate the low basal rate of MTV gene transcription; surprisingly, hormonal regulation of tyrosine aminotransferase activity is also defective in CR1, whereas several other cellular responses to dexamethasone are normal. In the second class of mutants, represented by CR4, dexamethasone stimulates synthesis of MTV transcripts indistinguishable from those produced in M1.54, but only the constitutive cell surface viral glycoprotein is expressed. Thus, these mutants define two distinct and novel aspects of glucocorticoid regulated gene expression in HTC cells: CR4 contains a defect in a hormone inducible protein maturation pathway that acts on specific viral (and presumably cellular) precursor polypeptides, whereas the lesion in CR1 appears to affect the expression of a subset of the gene products normally under glucocorticoid control in M1.54.

scholarly journals Exocytosis of Alphaherpesvirus Virions, Light Particles, and Glycoproteins Uses Constitutive Secretory Mechanisms

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Ian B. Hogue ◽  
Julian Scherer ◽  
Lynn W. Enquist
Keyword(s):  
Plasma Membrane ◽  
Virus Particle ◽  
Immune Evasion ◽  
Host Cells ◽  
Type I ◽  
Rab Gtpases ◽  
Viral Glycoprotein ◽  
Particle Assembly ◽  
Viral Glycoproteins ◽  
Light Particles

ABSTRACTMany molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based ontotalinternalreflectionfluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway.IMPORTANCEThe alphaherpesviruses, including the important human pathogens herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), are among the few viruses that have evolved to exploit the mammalian nervous system. These viruses typically cause mild recurrent herpetic or zosteriform lesions but can also cause debilitating herpes encephalitis, more frequently in very young, old, immunocompromised, or nonnatural hosts. Importantly, many of the molecular and cellular mechanisms of viral assembly and egress remain unclear. This study addresses the trafficking of viral glycoproteins to the plasma membrane, exocytosis of light particles, and exocytosis of virions. Trafficking of glycoproteins affects immune evasion and pathogenesis and may precede virus particle assembly. The release of light particles may also contribute to immune evasion and pathogenesis. Finally, exocytosis of virions is important to understand, as this final step in the virus replication cycle produces infectious extracellular particles capable of spreading to the next round of host cells.

Terminal α2-6 Linked Sialic Acid Expression On VWF Specifically Enhances Proteolysis by ADAMTS13.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 30-30
Author(s):  
Rachel Therese McGrath ◽  
Thomas A J McKinnon ◽  
Barry Byrne ◽  
Richard O'Kennedy ◽  
Michael Laffan ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Blood Group ◽  
Conflicts Of Interest ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Specific Effect ◽  
Cysteine Proteases ◽  
Binding Activity ◽  
Collagen Binding ◽  
Sodium Metaperiodate

Abstract Abstract 30 VWF is a large plasma sialoglycoprotein that mediates platelet tethering at sites of vascular injury. VWF function is dependent upon VWF multimeric composition, which is regulated in plasma by ADAMTS13. ABO(H) blood group determinants expressed on VWF N-linked glycans significantly influence susceptibility to ADAMTS13 proteolysis. In this study, we investigated whether terminal sialic acid residues expressed on the N- and O-linked glycans of VWF may also regulate proteolysis by ADAMTS13. VWF was initially purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF sialylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. The rate of glycosidase-treated VWF proteolysis by ADAMTS13 was determined by incubation with recombinant ADAMTS13 and subsequent measurement of residual VWF collagen binding activity. Complete VWF deglycosylation has been shown to enhance the rate of proteolysis by ADAMTS13. In contrast, enzymatic desialylation of VWF by α2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired the rate of ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to only 50±14% by ADAMTS13, compared to 11±7% for untreated VWF (p<0.01) at the same time point. Despite this, Neu-VWF exhibited increased susceptibility to proteases other than ADAMTS13 (trypsin, chymotrypsin and cathepsin B; all p<0.05). VWF sialylation is therefore a specific enhancer of ADAMTS13-mediated proteolysis. Consequently, quantification and molecular distribution of VWF sialylation was examined by sequential digestion and HPLC analysis. Total sialic acid expression on pdVWF was 167nmol/mg, of which the majority (133.4nmol/mg or 80.1%) was present on its N-linked glycan chains. Interestingly, despite the resistance to ADAMTS13 proteolysis observed upon complete desialylation, digestion of pdVWF with α2-3 neuraminidase to remove predominantly O-linked sialic acid did not influence the rate of ADAMTS13 proteolysis. Previous studies have demonstrated that VWF expressing different blood groups exhibit altered rates of proteolysis by ADAMTS13 (O ≥ B > A ≥ AB). Since α2-6 linked sialic acid and ABO(H) determinants are both expressed as terminal antigens on VWF N-linked glycans, the effect of desialylation upon blood group-specific VWF proteolysis by ADAMTS13 was determined. As expected, untreated group O VWF was cleaved significantly faster than group AB-VWF (p<0.05). However, the ability of ABO blood group to regulate ADAMTS13 proteolysis was completely ablated upon VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. This indicates that VWF sialylation constitutes a more important determinant of susceptibility to ADAMTS13 proteolysis than ABO(H) expression. Sialic acid can mediate protein-protein interactions through either conformational and/or charge-mediated mechanisms. Despite this, sodium metaperiodate treatment of pdVWF to remove sialic acid anionic charge did not influence the rate of proteolysis by ADAMTS13. In contrast, the ability of sialic acid to specifically enhance ADAMTS13 proteolysis of VWF was significantly attenuated at high urea concentrations (≥2M), supporting the hypothesis that VWF sialylation enhances proteolysis by ADAMTS13 by promoting a ADAMTS13-specific permissive conformation. These novel data demonstrate that although sialic acid protects VWF against proteolysis by serine and cysteine proteases, it also specifically enhances susceptibility to proteolysis by ADAMTS13. Moreover, the magnitude of this sialic acid-specific effect on VWF proteolysis by ADAMTS13 is more marked than that attributable to N-linked ABO(H) blood group antigen expression. Therefore, quantitative variation in VWF sialylation represents a key regulator of VWF multimeric composition, and as such, is likely to be of clear patho-physiological significance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Structure of the infectious salmon anemia virus receptor complex illustrates a unique binding strategy for attachment

2017 ◽  
Vol 114 (14) ◽  
pp. E2929-E2936 ◽  
Author(s):  
Jonathan D. Cook ◽  
Azmiri Sultana ◽  
Jeffrey E. Lee
Keyword(s):  
Viral Entry ◽  
Vaccine Development ◽  
Influenza Viruses ◽  
Infectious Salmon Anemia Virus ◽  
Viral Glycoprotein ◽  
Distinctive Features ◽  
Infectious Salmon Anemia ◽  
Resolution Model ◽  
Salmon Anemia Virus ◽  
Anemia Virus

Orthomyxoviruses are an important family of RNA viruses, which include the various influenza viruses. Despite global efforts to eradicate orthomyxoviral pathogens, these infections remain pervasive. One such orthomyxovirus, infectious salmon anemia virus (ISAV), spreads easily throughout farmed and wild salmonids, constituting a significant economic burden. ISAV entry requires the interplay of the virion-attached hemagglutinin-esterase and fusion glycoproteins. Preventing infections will rely on improved understanding of ISAV entry. Here, we present the crystal structures of ISAV hemagglutinin-esterase unbound and complexed with receptor. Several distinctive features observed in ISAV HE are not seen in any other viral glycoprotein. The structures reveal a unique mode of receptor binding that is dependent on the oligomeric assembly of hemagglutinin-esterase. Importantly, ISAV hemagglutinin-esterase receptor engagement does not initiate conformational rearrangements, suggesting a distinct viral entry mechanism. This work improves our understanding of ISAV pathogenesis and expands our knowledge on the overall diversity of viral glycoprotein-mediated entry mechanisms. Finally, it provides an atomic-resolution model of the primary neutralizing antigen critical for vaccine development.

scholarly journals Drosophila laminin: characterization and localization.

1987 ◽  
Vol 105 (5) ◽  
pp. 2383-2391 ◽  
Author(s):  
L I Fessler ◽  
A G Campbell ◽  
K G Duncan ◽  
J H Fessler
Keyword(s):  
Basement Membrane ◽  
Polyclonal Antibodies ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Basement Membranes ◽  
Velocity Sedimentation ◽  
Internal Organs ◽  
Differentiated Cells ◽  
Basement Membrane Component ◽  
Molecular Masses

Drosophila laminin was isolated from the medium of Drosophila Kc cell cultures. It was purified by velocity sedimentation, gel filtration, and chromatography. Drosophila laminin is a disulfide-linked molecule consisting of three chains with apparent molecular masses of 400, 215, and 185 kD. In electron micrographs, it has the cross-shaped appearance with globular domains characteristic of vertebrate laminin with closely similar dimensions. The amino acid composition and lectin-binding properties of Drosophila laminin are given. Polyclonal antibodies to Drosophila laminin were prepared and their specificity was established. In developing embryos immunofluorescence staining was detected between 6 and 8 h of development; and in sections of 8-9-h and older embryos immunostaining was seen at sites where basement membranes are present surrounding internal organs, muscles, underlying the hypodermal epithelium, and in the nervous system. Basement membrane staining was also seen in larva and adults. Cells from Drosophila embryos dissociated at the cellular blastoderm stage were grown in culture and some specific, differentiated cells synthesized laminin after several hours of culture as shown by immunofluorescence. The significance of the evolutionary conservation of the structure of this basement membrane component is discussed.

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