Structural analysis and lipid-binding properties of recombinant human surfactant protein a derived from one or both genes

Surfactant protein A (SP-A) constitutes an important part of the innate immune defense in the lung. In humans there are two functional genes (SP-A1 and SP-A2). The functional importance of having two distinct chain types in human SP-A is undefined. Amino acid substitutions in the primary structure of the protein may have effects on structural stability or on activity. To address this issue, SP-A1, SP-A2, and coexpressed SP-A1/SP-A2 variants were in vitro expressed in insect cells, purified, and used for study. We found the following: (1) Human SP-A variants expressed in insect cells, derived from one gene (SP-A1 or SP-A2) or both genes, differ in the relative extent and heterogeneity of oligomerization. SP-A1 and SP-A2 exist in small oligomeric forms, whereas coexpressed SP-A1/SP-A2 products favor the formation of larger oligomers. (2) Circular dichroic and fluorescence spectroscopic studies identified structural differences between SP-A variants in the collagen domain, with SP-A2 being more stable than SP-A1 but not in the calcium binding region. Recombinant human SP-A variants expressed in insect cells exhibit a lower melting temperature compared to native human SP-A. Oligomerization does not increase the thermal stability of the collagen domain of coexpressed SP-A1/SP-A2. (3) The ability of SP-A to undergo self-aggregation and induce phospholipid and bacterial lipopolysaccharide aggregation is greater for SP-A2 than for coexpressed SP-A1/SP-A2, which in turn is greater than that observed for SP-A1. The presence of SP-A1 polypeptide chains in coexpressed products modulates functional capabilities of SP-A, which depend on both the collagen and globular domains.     

Domains of surfactant protein A that affect protein oligomerization, lipid structure and surface tension

Surfactant protein A (SP-A) is an abundant protein found in pulmonary surfactant which has been reported to have multiple functions. In this review, we focus on the structural importance of each domain of SP-A in the functions of protein oligomerization, the structural organization of lipids and the surface-active properties of surfactant, with an emphasis on ultrastructural analyses. The N-terminal domain of SP-A is required for disulfide-dependent protein oligomerization, and for binding and aggregation of phospholipids, but there is no evidence that this domain directly interacts with lipid membranes. The collagen-like domain is important for the stability and oligomerization of SP-A. It also contributes shape and dimension to the molecule, and appears to determine membrane spacing in lipid aggregates such as common myelin and tubular myelin. The neck domain of SP-A is primarily involved in protein trimerization, which is critical for many protein functions, but it does not appear to be directly involved in lipid interactions. The globular C-terminal domain of SP-A clearly plays a central role in lipid binding, and in more complex functions such as the formation and/or stabilization of curved membranes. In recent work, we have determined that the maintenance of low surface tension of surfactant in the presence of serum protein inhibitors requires cooperative interactions between the C-terminal and N-terminal domains of the molecule. This effect of SP-A requires a high degree of oligomeric assembly of the protein, and may be mediated by the activity of the protein to alter the form or physical state of surfactant lipid aggregates.     

Role of the degree of oligomerization in the structure and function of human surfactant protein A

The role of the degree of oligomerization in the structure and function of human surfactant protein A (SP-A) was investigated using a human SP-A1 mutant (SP-A1(DeltaAVC,C6S)), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys(6) and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys(6) mutant lacked the NH(2)-terminal Ala(-3)-Val(-2)-Cys(-1) (DeltaAVC) extension present in some SP-A1 isoforms. SP-A1(DeltaAVC,C6S) was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1(DeltaAVC,C6S) was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated from human lung lavages. SP-A1(DeltaAVC,C6S) showed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin degradation. The T(m) was 32.7 degrees C for SP-A1(DeltaAVC,C6S) and 44.5 degrees C for SP-A1. Although SP-A1(DeltaAVC,C6S) was capable of binding to calcium, rough lipopolysaccharide, and phospholipid vesicles, this mutant was unable to induce rough lipopolysaccharide and phospholipid vesicle aggregation, to enhance the interfacial adsorption of SP-B/SP-C-surfactant membranes, and to undergo self-association in the presence of Ca(2+). On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1(DeltaAVC,C6S) to inhibit the production of tumor necrosis factor-alpha by macrophage-like U937 cells stimulated with either smooth or rough lipopolysaccharide. We conclude that supratrimeric assembly of human SP-A is essential for collagen triple helix stability at physiological temperatures, protection against proteases, protein self-association, and SP-A-induced ligand aggregation. The supratrimeric assembly is not essential for the binding of SP-A to ligands and anti-inflammatory effects of SP-A.     

Mechanism of stabilization of a bacterial collagen triple helix in the absence of hydroxyproline

The Streptococcus pyogenes cell-surface protein Scl2 contains a globular N-terminal domain and a collagen-like domain, (Gly-Xaa-X'aa)(79), which forms a triple helix with a thermal stability close to that seen for mammalian collagens. Hyp is a major contributor to triple-helix stability in animal collagens, but is not present in bacteria, which lack prolyl hydroxylase. To explore the basis of bacterial collagen triple-helix stability in the absence of Hyp, biophysical studies were carried out on recombinant Scl2 protein, the isolated collagen-like domain from Scl2, and a set of peptides modeling the Scl2 highly charged repetitive (Gly-Xaa-X'aa)(n) sequences. At pH 7, CD spectroscopy, dynamic light scattering, and differential scanning calorimetry of the Scl2 protein all showed a very sharp thermal transition near 36 degrees C, indicating a highly cooperative unfolding of both the globular and triple-helix domains. The collagen-like domain isolated by trypsin digestion showed a sharp transition at the same temperature, with an enthalpy of 12.5 kJ/mol of tripeptide. At low pH, Scl2 and its isolated collagen-like domain showed substantial destabilization from the neutral pH value, with two thermal transitions at 24 and 27 degrees C. A similar destabilization at low pH was seen for Scl2 charged model peptides, and the degree of destabilization was consistent with the strong pH dependence arising from the GKD tripeptide unit. The Scl2 protein contained twice as much charge as human fibril-forming collagens, and the degree of electrostatic stabilization observed for Scl2 was similar to the contribution Hyp makes to the stability of mammalian collagens. The high enthalpic contribution to the stability of the Scl2 collagenous domain supports the presence of a hydration network in the absence of Hyp.     

Three-dimensional structure of rat surfactant protein A trimers in association with phospholipid monolayers

Surfactant protein A (SP-A) is a C-type lectin found primarily in the lung and plays a role in innate immunity and the maintenance of surfactant integrity. To determine the three-dimensional (3D) structure of SP-A in association with a lipid ligand, we have used single particle electron crystallography and computational 3D reconstruction in combination with molecular modeling. Recombinant rat SP-A, containing a deletion of the collagen-like domain, was incubated with dipalmitoylphosphatidylcholine:egg phosphatidylcholine (1:1, wt/wt) lipid monolayers in the presence of calcium, negatively stained, and examined by transmission electron microscopy. Images of SP-A-lipid complexes with different angular orientations were used to reconstruct the 3D structure of the protein. These results showed that SP-A subunits readily formed trimers and interacted with lipid monolayers exclusively via the globular domains. A homology-based molecular model of SP-A was generated and fitted into the electron density map of the protein. The plane of the putative lipid-protein interface was relatively flat and perpendicular to the hydrophobic neck region, and the cleft region in the middle of the trimer had no apparent charge clusters. Amino acid residues that are known to affect lipid interactions, Glu(195) and Arg(197), were located at the protein-lipid interface. The molecular model indicated that the hydrophobic neck region of the SP-A did not interact with lipid monolayers but was instead involved in intratrimeric subunit interactions. The glycosylation site of SP-A was located at the side of each subunit, suggesting that the covalently linked carbohydrate moiety probably occupies the spaces between the adjacent globular domains, a location that would not sterically interfere with ligand binding.     

Pulmonary collectins modulate strain-specific influenza a virus infection and host responses

Collectins are secreted collagen-like lectins that bind, agglutinate, and neutralize influenza A virus (IAV) in vitro. Surfactant proteins A and D (SP-A and SP-D) are collectins expressed in the airway and alveolar epithelium and could have a role in the regulation of IAV infection in vivo. Previous studies have shown that binding of SP-D to IAV is dependent on the glycosylation of specific sites on the HA1 domain of hemagglutinin on the surface of IAV, while the binding of SP-A to the HA1 domain is dependent on the glycosylation of the carbohydrate recognition domain of SP-A. Here, using SP-A and SP-D gene-targeted mice on a common C57BL6 background, we report that viral replication and the host response as measured by weight loss, neutrophil influx into the lung, and local cytokine release are regulated by SP-D but not SP-A when the IAV is glycosylated at a specific site (N165) on the HA1 domain. SP-D does not protect against IAV infection with a strain lacking glycosylation at N165. With the exception of a small difference on day 2 after infection with X-79, we did not find any significant difference in viral load in SP-A(-/-) mice with either IAV strain, although small differences in the cytokine responses to IAV were detected in SP-A(-/-) mice. Mice deficient in both SP-A and SP-D responded to IAV similarly to mice deficient in SP-D alone. Since most strains of IAV currently circulating are glycosylated at N165, SP-D may play a role in protection from IAV infection.     

The role of glycosylation and domain interactions in the thermal stability of human angiotensin-converting enzyme

Abstract The N and C domains of somatic angiotensin-converting enzyme (sACE) differ in terms of their substrate specificity, inhibitor profiling, chloride dependency and thermal stability. The C domain is thermally less stable than sACE or the N domain. Since both domains are heavily glycosylated, the effect of glycosylation on their thermal stability was investigated by assessing their catalytic and physicochemical properties. Testis ACE (tACE) expressed in mammalian cells, mammalian cells in the presence of a glucosidase inhibitor and insect cells yielded proteins with altered catalytic and physicochemical properties, indicating that the more complex glycans confer greater thermal stabilization. Furthermore, a decrease in tACE and N-domain N-glycans using site-directed mutagenesis decreased their thermal stability, suggesting that certain N-glycans have an important effect on the protein's thermodynamic properties. Evaluation of the thermal stability of sACE domain swopover and domain duplication mutants, together with sACE expressed in insect cells, showed that the C domain contained in sACE is less dependent on glycosylation for thermal stabilization than a single C domain, indicating that stabilizing interactions between the two domains contribute to the thermal stability of sACE and are decreased in a C-domain-duplicating mutant.     

Roles of collagenous domain and oligosaccharide moiety of pulmonary surfactant protein A in interactions with phospholipids.

Pulmonary surfactant protein A (SP-A), a main component of lung-specific lipid-protein complex (pulmonary surfactant), is characterized by a collagen-like sequence in its amino terminal half and by N-linked glycosylation. The structural characteristics necessary for the various functions of SP-A are not yet completely understood. In the present study we examined the roles of the oligosaccharide moiety of SP-A and its collagenous domain in causing the aggregation of phospholipid liposomes and enhancing the uptake of phospholipids by type II cells. SP-A in the deglycosylated form increased turbidity, measured to evaluate liposome aggregation, to some extent at 400 nm, but this ability of the deglycosylated protein appeared to be less than that of control SP-A. The collagenase-resistant fragment of SP-A completely failed to aggregate phospholipid liposomes. Deglycosylated SP-A was able to enhance the uptake of phospholipids by type II cells, whereas removal of the collagenous domain of SP-A resulted in the loss of the ability to enhance phospholipid uptake.     

Aspects of secondary and quaternary structure of surfactant protein A from canine lung

The results of a large number of studies indicate that pulmonary surfactant contains a unique protein whose principal isoform has a molecular weight of about 30,000, and whose presence in surfactant is associated with important metabolic and physicochemical properties. This protein, SP-A, as isolated from canine surfactant, contains a domain of 24 repeating triplets of Gly-X-Y, similar to that found in collagens. These studies were undertaken to determine whether SP-A forms a collagen-like triple helix when in solution, and to describe certain aspects of its size and shape. Our experiments were done on SP-A extracted by two different methods from canine surfactant, and on SP-A produced by molecular cloning. The results from all three preparations were similar. The circular dichroism of the complete protein was characterized by a relatively large negative ellipticity at 205 nm, with a negative shoulder ranging from 215 to 230 nm. There was no positive ellipticity, and the spectrum was not characteristic of collagen. Trypsin hydrolysis resulted in a fragment with peak negative ellipticity at about 200 nm, without the negative shoulder. Further hydrolysis of this fragment with pepsin resulted in a CD spectrum similar to that of collagen. The spectrum of the collagen-like fragment was reversibly sensitive to heating to 50 degrees C, and was irreversibly lost after treatment with bacterial collagenase. SP-A migrated on molecular sieving gels with an equivalent Stokes radius of 110 to 120 A, and had a sedimentation coefficient of 14 S. Using these data we calculate a molecular weight of about 700,000. The hydrodynamic characteristics can be approximated as a prolate ellipsoid of revolution having an axial ratio of about 20. We conclude that SP-A aggregates into a complex of 18 monomers, which may form six triple-helices. The shape of the complex is considerably more globular than collagen and is not consistent with end-to-end binding of the helices to form fibrous structures.     

Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1-mediated phagocytosis

C1q, a subunit of the first component (C1) of the classical complement pathway, and the pulmonary surfactant protein SP-A are structurally homologous molecules, each having an extended collagen-like domain contiguous with a non-collagenous domain. It is the collagen-like region of C1q that binds to mononuclear phagocytes and mediates the enhancement of phagocytosis of opsonized particles by these cells. Because SP-A enhances the endocytosis of phospholipids by alveolar type II cells and alveolar macrophages, we examined whether these two molecules were functionally interchangeable. The phagocytosis of sheep erythrocytes opsonized with IgG or with IgM and complement was enhanced by the adherence of monocytes or macrophages, respectively, to SP-A. The enhanced response was dependent on the concentration of SP-A used for coating the surfaces, similar to that seen when monocytes were adhered to C1q-coated surfaces. Both the percentage of cells ingesting the opsonized targets and the number of targets ingested per cell increased with increasing concentrations of SP-A. No such enhancement was seen with cells adhered to albumin, iron-saturated transferrin, or uncoated surfaces. However, SP-A did not substitute for C1q in the formation of hemolytically active C1. C1q did not stimulate lipid uptake by alveolar type II cells or alveolar macrophages and had only a slight inhibitory effect on the binding of SP-A to alveolar type II cells. Thus, these results suggested that a function which requires interactions of both the collagenous and the non-collagenous regions (i.e. initiation of the classic complement cascade) could not be mimicked by a protein sharing structural macromolecular similarity but lacking sequence homology in the non-collagen-like region. However, SP-A could substitute for C1q in stimulating a function previously shown to be mediated by the collagen-like domains of the C1q molecule.     

A novel supersecondary structure in globular proteins comprising the collagen-like helix and beta-turn

A structure consisting of the polyproline-II or collagen-like helix immediately succeeded by a beta-turn is seen in several synthetic peptides and has been suggested to be the conformational requirement for proline hydroxylation in nascent procollagen. Using a simple algorithm for detecting secondary structures, we have analysed crystal structure data on 40 globular proteins and have found eight examples of the collagen-helix + beta-turn supersecondary structure in 15 proteins that contain the collagen-like helical segments.     

Structural characterisation of human proteinosis surfactant protein A

Human surfactant protein-A (SP-A) has been purified from a proteinosis patient and characterised by a combination of automated Edman degradation and mass spectrometry. The complete protein sequence was characterised. The major part of SP-A was shown to consist of SP-A2 gene product, and only a small amount of SP-A1 gene product was shown to be present. A cysteine extension to the N-terminal was indicated by sequence data, but was not definitely proven. All proline residues in the Y position of Gly-X-Y in the collagen-like region were at least partially modified to hydroxy-proline, but no lysine residues were found to be modified. A complex N-linked glycosylation was found on Asn-187 showing great heterogeneity as variants from a mono-antennary to penta-antennary glycosylation with varying amounts of attached pentose were identified. The disulfide bridges in the carbohydrate recognition domain were identified to be in the 1-4, 2-3 pattern common for collectins. Interchain disulfide bridges were discovered between two Cys-48 residues and cysteine residues in the N-terminal region. However, the exact disulfide bridge connections within the bouquet-like ultrastructure could not be established.     

Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.     

The Crystal Structure of the Streptococcal Collagen-like Protein 2 Globular Domain from Invasive M3-type Group A Streptococcus Shows Significant Similarity to Immunomodulatory HIV Protein gp41

The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.     

Glycosylation of a disintegrin and metalloprotease 17 affects its activity and inhibition

ADAM17 (a disintegrin and metalloprotease 17) is believed to be a tractable target in various diseases, including cancer and rheumatoid arthritis; however, it is not known whether glycosylation of ADAM17 expressed in healthy cells differs from that found in diseased tissue and, if so, whether glycosylation affects inhibitor binding. We expressed human ADAM17 in mammalian and insect cells and compared their glycosylation, substrate kinetics, and inhibition profiles. We found that ADAM17 expressed in mammalian cells was more heavily glycosylated than its insect-expressed analog. To determine whether differential glycosylation modulates enzymatic activity, we performed kinetic studies with both ADAM17 analogs and various TNFα-based substrates. The mammalian form of ADAM17 exhibited 10- to 30-fold lower k_cat values than the insect analog, while the K_M was unaffected, suggesting that glycosylation of ADAM17 can potentially play a role in regulating enzyme activity in vivo. Finally, we tested ADAM17 forms for inhibition by several well-characterized inhibitors. Active-site zinc-binding small molecules did not exhibit differences between the two ADAM17 analogs, while a non-zinc-binding exosite inhibitor of ADAM17 showed significantly lower potency toward the mammalian-expressed analog. These results suggest that glycosylation of ADAM17 can affect cell signaling in disease and might provide opportunities for therapeutic intervention using exosite inhibitors.     

Role of N-linked glycans of HCV glycoprotein E1 in folding of structural proteins and formation of viral particles

Envelope proteins E1 and E2 of the hepatitis C virus (HCV) play a major role in the life cycle of a virus. These proteins are the main components of the virion and are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation, which is supposed to play a role in their stability, in the assembly of the functional glycoprotein heterodimer, in protein folding, and in viral entry. The effects of N-linked glycosylation of HCV protein E1 on the assembly of structural proteins were studied using site-directed mutagenesis in a model system of Sf9 insect cells producing three viral structural proteins with the formation of virus-like particles due to the baculovirus expression system. The removal of individual N-glycosylation sites in HCV protein E1 did not affect the efficiency of its expression in insect Sf9 cells. The electrophoretic mobility of E1 increased with a decreasing number of N-glycosylation sites. The destruction of E1 glycosylation sites N1 or N5 influenced the assembly of the noncovalent E1E2 glycoprotein heterodimer, which is the prototype of the natural complex within the HCV virion. It was also shown that the lack of glycans at E1 sites N1 and N5 significantly reduced the efficiency of E1 expression in mammalian HEK293 T cells.     

Human SP-A genetic variants and bleomycin-induced cytokine production by THP-1 cells: effect of ozone-induced SP-A oxidation

Surfactant protein A (SP-A) plays a role in innate host defense. Human SP-A is encoded by two functional genes (SP-A1 and SP-A2), and several alleles have been characterized for each gene. We assessed the effect of in vitro expressed human SP-A genetic variants, on TNF-alpha and IL-8 production by THP-1 cells in the presence of bleomycin, either before or after ozone-induced oxidation of the variants. The oligomerization of SP-A variants was also examined. We found 1) cytokine levels induced by SP-A2 (1A, 1A(0)) were significantly higher than those by SP-A1 (6A(2), 6A(4)) in the presence of bleomycin. 2) In the presence of bleomycin, ozone-induced oxidation significantly decreased the ability of 1A and 1A/6A(4), but not of 6A(4), to stimulate TNF-alpha production. 3) The synergistic effect of bleomycin/SP-A, either before or after oxidation, can be inhibited to the level of bleomycin alone by surfactant lipids. 4) Differences in oligomerization were also observed between SP-A1 and SP-A2. The results indicate that differences among SP-A variants may partly explain the individual variability of pulmonary complications observed during bleomycin chemotherapy and/or in an environment that may promote protein oxidation.     

Thermal stability and DPPC/Ca2+ interactions of pulmonary surfactant SP-A from bulk-phase and monolayer IR spectroscopy.

Surfactant protein A (SP-A), the most abundant pulmonary surfactant protein, is implicated in multiple biological functions including surfactant homeostasis, biophysical activity, and host defense. SP-A forms ternary complexes with lipids and Ca2+ which are important for protein function. The current study uses infrared (IR) transmission spectroscopy to investigate the bulk-phase interaction between SP-A, 1,2-dipalmitoylphosphatidylcholine (DPPC), and Ca2+ ions along with IR reflection-absorption spectroscopy (IRRAS) to examine protein secondary structure and lipid orientational order in monolayer films in situ at the air/water interface. The amide I contour of SP-A reveals two features at 1653 and 1636 cm(-1) arising from the collagen-like domain and a broad feature at 1645 cm(-1) suggested to arise from the carbohydrate recognition domain (CRD). SP-A secondary structure is unchanged in lipid monolayers. Thermal denaturation of SP-A in the presence of either DPPC or Ca2+ ion reveals a sequence of events involving the initial melting of the collagen-like region, followed by formation of intermolecular extended forms. Interestingly, these spectral changes were inhibited in the ternary system, showing that the combined presence of both DPPC and Ca2+ confers a remarkable thermal stability upon SP-A. The ternary interaction was revealed by the enhanced intensity of the asymmetric carboxylate stretching vibration. The IRRAS measurements indicated that incorporation of SP-A into preformed DPPC monolayers at a surface pressure of 10 mN/m induced a decrease in the average acyl chain tilt angle from 35 degrees to 28 degrees. In contrast, little change in chain tilt was observed at surface pressures of 25 or 40 mN/m. These results are consistent with and extend the fluorescence microscopy studies of Keough and co-workers [Ruano, M. L. F., et al. (1998) Biophys. J. 74, 1101-1109] in which SP-A was suggested to accumulate at the liquid-expanded/liquid-condensed boundary. Overall these experiments reveal the remarkable stability of SP-A in diverse, biologically relevant environments.     

Mutations of rat surfactant protein A have distinct effects on its glycosylation,secretion, aggregation and degradation

Aims

Surfactant protein A (SP-A) plays critical roles in the innate immune system and surfactant homeostasis of the lung. Mutations in SP-A2 of the carbohydrate recognition domain (CRD) impair its glycosylation and are associated with pulmonary fibrosis in humans. We aim to examine how mutations in SP-A that impair its glycosylation affect its biological properties and lead to disease.

Main methods

We generated rat SP-A constructs with two types of mutations that impair its glycosylation: N-glycosylation site mutations (N21T, N207S and N21T/N207S) and disease-associated CRD mutations (G231V, F198S). We transfected these constructs into Chinese hamster ovary (CHO)-K1 cells and assessed biochemical differences in cellular and secreted wild-type and mutant SP-As by western blot, immunofluorescence, and sensitivity to enzymatic digestion.

Key findings

Mutations of the CRD completely impaired SP-A secretion, whereas mutations of N-glycosylation sites had little effect. Both types of mutations formed nonidet p-40 (NP-40) insoluble aggregates, but the aggregates only from CRD mutations could be partially rescued by a chemical chaperone, 4-phenylbutyrate acid (4-PBA). The majority of CRD mutant SP-A was retained in the endoplasmic reticulum. Moreover, both types of mutations reduced SP-A stability, with CRD mutant SP-A being more sensitive to chymotrypsin digestion. Both types of soluble mutant SP-A could be degraded by the proteasome pathway, while insoluble aggregates could be additionally degraded by the lysosomal pathway.

Significance

Our data provide evidence that the differential glycosylation of SP-A may play distinct roles in SP-A secretion, aggregation and degradation which may contribute to familial pulmonary fibrosis caused by SP-A2 mutations.     

Collagen-like proteins in pathogenic E. coli strains

The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.