Glycosylation and secretion of surfactant-associated glycoprotein A

Synthesis of glycoprotein A, the major surfactant-associated protein, was demonstrated in Type II epithelial cells isolated from rat lung. Predominant, secreted forms migrated as glycoproteins with asparagine-linked, complex-type oligosaccharides (32,000-36,000 daltons, pI 4.2-4.8). Primary in vitro translation products of the glycoprotein migrated as five distinct proteins of approximately 26,000 daltons which were processed by pancreatic microsomal membranes in vitro to 30,000-34,000-dalton, endoglycosidase F-sensitive forms. These in vitro processed forms of glycoprotein A co-migrated with intracellular forms immunoprecipitated from [35S]methionine-labeled, Type II cells. Pulse-chase experiments with [35S]methionine-labeled cells demonstrated rapid synthesis of endoglycosidase H-sensitive precursors of 34,000 daltons, pI 4.7-4.8, which were neither secreted from Type II cells nor detected in surfactant from alveolar lavage. These high-mannose forms were slowly processed to more acidic, endoglycosidase H-resistant, neuraminidase-sensitive forms. At between 10 and 180 min, fully sialylated or other endoglycosidase H-resistant forms were a minor fraction of intracellular glycoprotein A. After 16 h, intracellular glycoproteins A were primarily present as endoglycosidase H-resistant forms. Secretion of mature, sialylated, glycoprotein A was first detected 1 h after labeling, and was also readily detected after 16-24 h chase period. Tunicamycin, which blocks N-linked protein glycosylation, resulted in synthesis of three major 26,000-dalton proteins which co-migrated with the nonglycosylated, surfactant-associated proteins A1 present in surfactant from alveolar lavage and with the major in vitro translation products of rat lung poly(A+) mRNA. Tunicamycin inhibited secretion of glycoprotein A. Swainsonine, which inhibits Golgi alpha-mannosidase II, completely inhibited synthesis of the fully sialylated molecule. Swainsonine produced forms of glycoprotein A which were both neuraminidase- and endoglycosidase H-sensitive and were readily secreted. Monensin, an ionophore that alters protein transport, markedly inhibited intracellular sialylation and secretion. These studies demonstrate that pulmonary Type II cells rapidly synthesize and process surfactant-associated glycoprotein A precursors to endoglycosidase H-sensitive forms, which are slowly sialylated prior to secretion.     

In vitro sulfation of pulmonary surfactant-associated protein-35

Surfactant-associated protein-35 consists of a group of phospholipid-associated proteins of 26-36 kDa isolated from pulmonary alveolar surfactant. In the rat, surfactant-associated protein-35 is synthesized from 26-kDa primary translation products which are cotranslationally acetylated and glycosylated to heterogeneous 30 and 34 kDa forms. High-mannose oligosaccharide-containing precursors of surfactant-associated protein-35 are processed in the rough endoplasmic reticulum and Golgi to complex-type oligosaccharides, resulting in a mature glycoprotein which exhibits extensive charge heterogeneity in two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis. Much of this charge heterogeneity is related to terminal sialylation of the two asparagine-linked oligosaccharides. In the present study, we report that surfactant-associated protein-35 is also sulfated. Sulfation of the 30 and 34 kDa forms of surfactant-associated protein-35 was clearly detected in primary cultures of rat Type II epithelial cells. These sulfated isoforms were sensitive to endoglycosidase F digestion, but resistant to neuraminidase, suggesting that sulfation occurred at oligosaccharide residues other than sialic acid. The lack of sulfation of the 26 kDa forms of surfactant-associated protein-35 and the resistance of the sulfated isoforms to endoglycosidase H digestion are consistent with Golgi-associated sulfation of the complex type oligosaccharides of surfactant-associated protein-35. Thus, sulfation is another component of the complex post-translational processing of surfactant-associated protein-35, which includes acetylation, hydroxylation, glycosylation, sialylation, sulfhydryl-dependent oligomerization and sulfation.     

Intracellular and oligomeric forms of surfactant-associated apolipoproteins(s) A in the rat

Sulfhydryl-dependent oligomeric forms of the surfactant-associated apolipoprotein(s) A, obtained from particulate preparations of adult rat lung lavage, were characterized by immunoblot analysis and by silver staining of proteins separated by one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Under non-reducing conditions, these proteins migrated as oligomers, Mr approx. 50-70, 115, 160 kDa and greater. The large oligomers were reduced to the apolipoprotein(s) A subunits by treatment with beta-mercaptoethanol; Mr 38 (A3), 32 (A2) and 26 kDa (A1), pI 4.2-4.8. Mr 50 kDa protein was composed of sulfhydryl-dependent homo-dimers of protein(s) A1 (Mr 26 kDa). 55 kDa protein was a hetero-dimer composed primarily of A1 and A2 (Mr 26 and 32 kDa). 62 kDa protein was composed of hetero-dimers of A3 and apolipoprotein A2 (Mr 38 and 32 kDa). 70 kDa protein was a homodimer composed of apolipoprotein A3 A3 (38 kDa). Larger molecular forms were composed primarily of 38 and 32 kDa and lesser amounts of 26 kDa. Treatment with endoglycosidase F reduced A2 and A3 to 26 kDa. Apolipoprotein A1 co-migrated with a protein of Mr 26 kDa immunoprecipitated from [35S]methionine-labelled Type II epithelial cells. Chymotryptic-tryptic peptide maps of apolipoproteins A1, A2 and A3 were identical, suggesting that apolipoproteins A3 and A2 arise through extensive glycosylation of apolipoprotein A1.     

Purification of canine surfactant-associated glycoproteins A. Identification of a collagenase-resistant domain

A procedure for purification of surfactant-associated glycoproteins A from canine surfactant was established utilizing preparative isoelectric focusing as a major purification step in absence of detergents. The proteins migrated as charge trains, isoelectric points 4.2-5.0. Unglycosylated forms of surfactant-associated protein A1 (26 kDa) and glycoproteins A2 and A3 (32-36 kDa) were identified by silver-staining and immunoblot analysis. These forms were demonstrated to be identical polypeptides by fingerprint analysis of 125I-labeled peptides generated by tryptic-chymotryptic digests of the iodinated proteins. Size and charge heterogeneity were related to varying amounts of N-linked complex carbohydrates, including sialic acid, which were sensitive to endoglycosidase F and neuraminidase but resistant to endoglycosidase H. A collagenase-sensitive region was demonstrated which was required for sulfhydryl-dependent oligomerization of the molecule. Collagenase treatment resulted in removal of approx. 10 kDa from the glycoprotein molecule. Collagenase-resistant fragments of 21-23 kDa migrated with carbohydrate-dependent size and charge heterogeneity and were reduced to 16 kDa by endoglycosidase F. Amino acid composition of the surfactant glycoproteins demonstrated high glycine content which was diminished after digestion with collagenase. Several glycine-rich tryptic peptides were isolated by reverse-phase chromatography. Partial sequence information shows Gly-X-Y repeat sequences containing hydroxyproline residues. The major canine surfactant-associated proteins, glycoproteins A contain complex-type N-linked carbohydrate. In addition, a separate collagen-like peptide domain is present and is required for sulfhydryl-dependent oligomerization.     

Identification of canine pulmonary surfactant-associated glycoprotein A precursors

Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000-28,000 daltons; glycoprotein A2, 32,000-34,000 daltons; and glycoprotein A3, 37,000-38,000 daltons; pH at isoelectric point (pI) 4.5-5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8-5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000-34,000 daltons, pI 4.8-5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000-28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.     

Structural relationships of the major glycoproteins from human alveolar proteinosis surfactant

Alveolar proteinosis is a disease characterized by accumulation of proteinaceous material in the alveolar space of the lung. Two major collagenase-sensitive polypeptides, alveolar proteinosis peptides of 34 kDa kilodaltons (APP-34) and of 62 kDa (APP-62), were isolated from bronchioalveolar lavage of patients with alveolar proteinosis. These proteins co-purified during fast-performance liquid chromatography (FPLC) chromatofocusing and were separated from each other by electroelution following SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of these proteins demonstrated that both shared antigenic sites with the normal human surfactant-associated protein of Mr 34,000 (SAP-34) using both polyclonal and monoclonal antibodies generated against SAP-34. Removal of asparagine-linked oligosaccharides from the 34 kDa and 62 kDa alveolar proteinosis proteins with endoglycosidase F resulted in polypeptides of 28 kDa from APP-34 and 56 kDa from APP-62. Amino acid analysis and tryptic peptide maps of the electroeluted APP-34 and APP-62 proteins were essentially identical and similar to that previously reported for human SAP-34, supporting the likely relationship of APP-34 and APP-62 as monomer and dimer of the normal SAP-34. APP-34 and APP-62 were both sensitive to bacterial collagenase, yielding collagenase-resistant fragments of 21 kDa, similar in migration and amino acid composition to the fragment generated by collagenase digestion of normal human SAP-34. High molecular weight aggregates of APP-34 and APP-62 were the result of sulfhydryl-dependent and non-sulfhydryl-dependent cross-linking. A domain in the C-terminal non-collagenous portion of the molecules which forms sulfhydryl-dependent oligomers was identified. The two major polypeptides accumulating in the airway of patients with alveolar proteinosis are monomeric (34 kDa) and dimeric (62 kDa) forms of the major surfactant-associated glycoprotein, SAP-34.     

Mapping of surface glycoproteins of Trypanosoma cruzi by two-dimensional electrophoresis. A correlation with the cell invasion capacity

The cell-surface iodinatable proteins of Trypanosoma cruzi have been analyzed by two-dimensional polyacrylamide gel electrophoresis under equilibrium conditions. Antigenic polypeptides were characterized after immunoprecipitation and glycoproteins were identified by means of lectin-affinity chromatography. Two glycoproteins, with affinity for concanavalin A, were found to be common to both infective (trypomastigote) and non-infective (epimastigote) forms: protein 1 (90 kDa, pI 5.5-6.5) and protein 2 (80 kDa, pI 5.3-6.3). In epimastigotes a specific concanavalin-A-binding surface glycoprotein (70 kDa, pI 5.5) was identified. Trypomastigote forms, on the other hand, presented several specific iodinatable surface components: glycoproteins 3(85 kDa, pI 5.5), 4 (85 kDa, pI 5.0), 6 (100 kDa, pI 6.5), 7 (120 kDa, pI 6.3), 8 (68 kDa, pI 6.7) and several minor high-molecular-mass acid proteins, all containing glucose and/or mannose, and glycoprotein 5 (85 kDa, pI 6.3-7.5), containing N-acetyl-D-glucosamine (Tc-85). Proteins 1, 2 and 5 were the only ones which gave clear evidence of charge heterogeneity. Most of the surface proteins of trypomastigote forms, the exception being proteins 3, 4 and 8, were removed by treatment with trypsin. This proteolytic treatment results in 90% inhibition of the in vitro vertebrate-cell-invasion capacity of the parasites. Upon reincubation in culture medium for 4 h, the trypsin-removed glycoproteins are again detected, an observation that correlates well with the recovery of the cell-penetration capacity observed in the same period.     

Differential effects of glucocorticoid on expression of surfactant proteins in a human lung adenocarcinoma cell line

Synthesis of pulmonary surfactant-associated glycoproteins of Mr 28,000-36,000 (SP-A) and Mr 42,000-46,000 (proSP-B) has been identified in a continuous cell line derived from a human lung adenocarcinoma. SP-A was detected by immunoblot analysis, ELISA assay and by [35S]methionine labelling of the cells. SP-A was secreted into the media as an endoglycosidase F sensitive glycoprotein which co-migrated with the isoforms of SP-A identified in human lavage fluid by 2D-IEF-SDS-PAGE. Hybridization of cellular RNA with SP-A-specific cDNA identified an abundant 2.2 kb mRNA species, identical to that observed in human lung. SP-A RNA and protein content were markedly inhibited by dexamethasone in a dose-dependent fashion. Under identical culture conditions, synthesis of a distinct surfactant protein, SP-B, was markedly stimulated by the glucocorticoid. The SP-B precursor was secreted into the media as heterogeneous Mr 42,000-46,000 protein, pI 4.6-5.1, and was sensitive to endoglycosidase F. Synthesis of proSP-B was enhanced by the glucocorticoid in a dose-dependent fashion and was associated with increased SP-B mRNA of 2.0 kb detected by Northern blot analysis. The cell line secreted proSP-B as Mr 42,000-46,000 glycosylated protein and did not process the precursor to the Mr 7000-8000 surfactant peptide. In summary, a human adenocarcinoma cell line has been identified which synthesizes and secretes two surfactant-associated proteins, SP-A and proSP-B. Glucocorticoid enhanced SP-B but inhibited SP-A expression in this cell line. The identification of a continuous cell line secreting surfactant proteins may be useful in the study of synthesis and secretion of these important proteins and for production of the proteins for clinical uses.     

Primary translation products, biosynthesis, and tissue specificity of the major surfactant protein in rat

Rat lung tissue was labeled with [35S]methionine and the major surfactant-associated proteins immunoprecipitated using a specific antiserum. The protein pattern obtained was very similar to that seen in rat bronchoalveolar lavage. Rat lung mRNA was subsequently translated in an in vitro rabbit reticulocyte system, and surfactant-associated protein-related polypeptides were immunoprecipitated. A 26-kDa polypeptide was identified and characterized as follows. (a) Unlabeled surfactant proteins added to the immunoprecipitation mixture completely inhibited its immunoprecipitation. (b) Two-dimensional gel electrophoresis of the 26-kDa protein resolved it into 3 isoforms. (c) Inclusion of dog pancreatic microsomes in the translation mixture resulted in the formation of two distinct higher molecular weight groups of isoforms, suggesting that the 26-kDa protein is destined to become a glycoprotein. Immunoprecipitation of [35S]methionine-labeled rat lung tissue proteins after tunicamycin treatment resulted in 3 isoforms, identical to the ones seen in the primary translation products. In addition, expression of the surfactant proteins appears specific to the lung.     

Immunologically related multimeric forms of 30–40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000–40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high molecular-weight proteins (> 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30–40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

Con A acceptor glycoproteins were analyzed by 2D-PAGE and 125I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)-transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 (MW 34 kDa, pI 5.1) was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21 (MW 21 kDa, pI 6.3), was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation.     

Purification and biochemical characterization of CP4 (SP-D), a collagenous surfactant-associated protein

CP4 is a collagenous glycoprotein (43 kDa, reduced) synthesized by rat type II pulmonary epithelial cells in primary culture (Persson et al., 1988). In order to better characterize this protein, CP4 was isolated from rat bronchoalveolar lavage and EDTA extracts of lung surfactant by adsorption to barium sulfate and elution with sodium citrate followed by reverse-phase HPLC. Amino acid analysis of purified CP4 demonstrated 4-hydroxyproline (Hyp), hydroxylysine (Hyl), and acid-labile components coeluting with Hyl glycosides. In addition, gas-phase amino-terminal microsequencing of two CP4 CNBr peptides demonstrated nonoverlapping collagenous sequences comprised of nine and six Gly-X-Y triplets, containing a total of four residues of Hyp and two of Hyl. There was less than 50% sequence homology of these peptides with the cDNA-derived sequence of the collagenous domain of rat SP-A. Two-dimensional IEF/SDS-PAGE resolved the protein into a charge train of basic isoforms (pI approximately 6-8), similar to those of newly synthesized CP4 and the class D surfactant proteins (Phelps & Taeusch, 1985). Gel filtration of nondenatured CP4 on 4% agarose showed a high apparent molecular mass complex comprised of disulfide-bonded trimers of the 43-kDa subunits. Antibodies to purified lavage CP4 showed specific binding to newly synthesized and surfactant-associated CP4. We propose that CP4 be designated "surfactant protein D" (SP-D) in accordance with an accepted nomenclature for surfactant-associated proteins.     

Proteomic analysis of human bronchoalveolar lavage fluid: expression profiling of surfactant-associated protein A isomers derived from human pulmonary alveolar proteinosis using immunoaffinity detection

Human bronchoalveolar lavage fluid (BALF) proteins from pulmonary alveolar proteinosis (PAP) obtained by washing the epithelial lining of the lung with phosphate-buffered saline, were separated using high resolution two-dimensional gel electrophoresis (2-DE) under denaturing and reducing conditions. By Western blotting, the proteins were transferred from polyacrylamide gel onto a chemical resilient membrane. The surfactant-associated protein A (SP-A) isomers were then identified with enhanced chemiluminescence detection (ECL) using antibody-antigen reaction. Some of the gels were treated with silver staining after 2-DE. The molecular masses of SP-A isomers in BALF from PAP ranged from 20.5 to 26, 26 to 32, and 32 to 42 kDa, respectively; and isoelectric points (pI) were in pH range of 4.5-5.4 under denaturing and reducing conditions. In the mass range of 20.5-26 kDa and pI of 4.5-5.4, there were five isomers, and in mass range of 26-32 kDa and pI of 4.5 to 5.4, there were at least eight isomers on the ECL detection film. However, in the mass range of 32-42 kDa and pI of 4.5-5.4, there were three isomers separated one from another but there was also a cluster of overlapping spots on the ECL detection film. Thus, this communication describes a characteristic 2-DE pattern of SP-A isomers in BALF from PAP as follows. (1) The five isomers of mass 20.5-26 kDa and pI of 4.5-5.4; (2) the eight isomers of mass 26-32 kDa and pI of 4.5-5.4; and (3) the three isomers of mass 32-42 kDa and pI of 4.5-5.4.     

Analysis of pulmonary surfactant apoproteins by isoelectric focusing

Apoproteins of Mr 38 000, 32 000 and 26 000 are found in surfactant isolated from rat lungs. The surfactant isolated from monkey lungs, on the other hand, contains the 38 kDa apoprotein and not the 32 and 26 kDa apoproteins. These preparations of pulmonary surfactant contain, in addition, several serum proteins. We have used a combination of salt- and sucrose-density gradient centrifugations to isolate and further purify surfactant from the washings of rat lungs. Thus, a preparation of pulmonary surfactant was obtained which contained exclusively the 38, 32, 26 and 10-12 kDa apoproteins, and which was rich in phosphatidylcholine and phosphatidylglycerol. Using an immunoassay and an immunoblotting technique, it was established that the 38, 32 and 26 kDa apoproteins are not serum proteins. The surfactant apoproteins of rat and monkey were further subjected to the high-resolution of isoelectric focusing. Thus, rat surfactant apoproteins resolved into 11 bands in the pH range 4.64-5.53. A second-dimensional electrophoresis in a sodium dodecyl sulfate system led to the migration of the 11 bands, separated by first-dimensional isoelectric focusing, into three distinct groups with apparent molecular weights of 38 000, 32 000 and 26 000, respectively. Upon isoelectric focusing, the apoproteins from monkey lung surfactant also separated into several bands in the pH range 5.18-5.82. After electrophoresis in the second dimension as above, these bands migrated as a single group with an apparent molecular weight of 38 000. Neuraminidase treatment of rat surfactant apoproteins, and subsequent IEF, led to the disappearance of several low-pI variants with a concomitant increase in the amounts of higher-pI variants. Thus, the sialic acid content of surfactant apoproteins accounts for, in large part, the observed charge heterogeneity.     

Immunologically related multimeric forms of 30-40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000-40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high-molecular-weight proteins (greater than 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30-40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

No evidence for vitamin K-dependent carboxylation of canine surfactant apoproteins, 28-36 kDa.

Recent research has shown that rat surfactant apoproteins (26-38 kDa) are vitamin K-dependent [Rannels, Gallaher, Wallin & Rannels (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5952-5956]. We have investigated the effect of the vitamin K antagonist warfarin on this family of apoproteins in surfactant from dog lung. Our data suggest that warfarin does not interfere with synthesis and secretion of these proteins into dog lung surfactant. Abnormal surfactant apoproteins, produced in response to warfarin treatment of the dog, were also not found in lung surfactant. 4-Carboxyglutamic acid analysis of purified dog apoproteins also failed to detect the vitamin K-modification. When vitamin K-dependent 14C labelling of precursors of vitamin K-dependent proteins was carried out, fluorography of these precursors, when electrophoresed into SDS/polyacrylamide gels, revealed 14C-labelled proteins of apparent molecular mass 74, 46, 42, 34, 31 and 23 kDa. Antibodies produced against purified dog surfactant apoproteins recognized precursors of the surfactant apoproteins in lung microsomes but did not recognize any 14C-labelled carboxylase substrates. These precursors appeared on immunoblots with apparent molecular mass 29, 32, 33 and 50 kDa. Our data suggest that there are significant differences between this class of surfactant apoproteins in the rat and the dog.     

alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins.

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.     

The transmembrane signaling pathway involved in directed movements of Chlamydomonas flagellar membrane glycoproteins involves the dephosphorylation of a 60-kD phosphoprotein that binds to the major flagellar membrane glycoprotein

Cross-linking of Chlamydomonas reinhardtii flagellar membrane glycoproteins results in the directed movements of these glycoproteins within the plane of the flagellar membrane. Three carbohydrate-binding reagents (FMG-1 monoclonal antibody, FMG-3 monoclonal antibody, concanvalin A) that induce flagellar membrane glycoprotein crosslinking and redistribution also induce the specific dephosphorylation of a 60- kD (pI 4.8-5.0) flagellar phosphoprotein (pp60) that is phosphorylated in vivo on serine. Ethanol treatment of live cells induces a similar specific dephosphorylation of pp60. Affinity adsorption of flagellar 32P-labeled membrane-matrix extracts with the FMG-1 monoclonal antibody and concanavalin A demonstrates that pp60 binds to the 350-kD class of flagellar membrane glycoproteins recognized by the FMG-1 monoclonal antibody. In vitro, protein phosphatase 2B (calcineurin) removes 60% of the 32P from pp60; this correlates well with previous observations that directed flagellar glycoprotein movements are dependent on micromolar calcium in the medium and are inhibited by calcium channel blockers and calmodulin antagonists. The data reported here are consistent with the dephosphorylation of pp60 being a step in the signaling pathway that couples flagellar membrane glycoprotein cross-linking to the directed movements of flagellar membrane glycoproteins.     

Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity.

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.