Deletion Mapping of N-terminal Domains of Surfactant Protein A

Abstract

The objective of the current study was to examine the functional importance of the N-terminal domains of surfactant protein A (SP-A) including the N-terminal segment from Asn1 to Ala7 (denoted domain 1), the N-terminal portion of the collagen domain from Gly8 to Gly44 (domain 2), and the C-terminal portion of the collagen-like domain from Gly45 to Pro80(domain 3). Wild type recombinant SP-A (SP-Ahyp; where hyp indicates hydroxyproline-deficient) and truncated mutant (TM) SP-As containing deletions of domain(s) 1 (TM1), 2 (TM2), 1 and 2 (TM1–2), and 1, 2, and 3 (TM1–2-3) were synthesized in insect cells and purified by mannose-Sepharose affinity chromatography. N-terminal disulfide-dependent dimerization was preserved at near wild type levels in the TM1–2 (at Cys−1) and TM2 proteins (at Cys−1 and Cys6), and to a lesser extent in TM1 (at Cys−1), but not in TM1–2-3. Cross-linking analyses demonstrated that the neck + CRD was sufficient for assembly of monomers into noncovalent trimers and that the N-terminal segment was required for the association of trimers to form higher oligomers. All TM proteins except TM1–2-3 bound to phospholipid, but only the N-terminal segment containing TM proteins aggregated phospholipid vesicles. The TM1, TM1–2, and TM2 but not the TM1–2-3 inhibited the secretion of surfactant from type II cells as effectively as SP-Ahyp, but the inhibitory activity of each mutant was blocked by excess α-methylmannoside and therefore nonspecific. TM1 and TM1–2-3 did not enhance the uptake of phospholipids by isolated type II cells, but the TM1–2 and TM2 had activities that were 72 and 83% of SP-Ahyp, respectively. We conclude the following for SP-A: 1) trimerization does not require the collagen-like region or interchain disulfide linkage; 2) the N-terminal portion of the collagen-like domain is required for specific inhibition of surfactant secretion but not for binding to liposomes or for enhanced uptake of phospholipids into type II cells; 3) N-terminal interchain disulfide linkage can functionally replace the N-terminal segment for lipid binding, receptor binding, and enhancement of lipid uptake; 4) the N-terminal segment is required for the association of trimeric subunits into higher oligomers, for phospholipid aggregation, and for specific inhibition of surfactant secretion and cannot be functionally replaced by disulfide linkage alone for these activities. The objective of the current study was to examine the functional importance of the N-terminal domains of surfactant protein A (SP-A) including the N-terminal segment from Asn1 to Ala7 (denoted domain 1), the N-terminal portion of the collagen domain from Gly8 to Gly44 (domain 2), and the C-terminal portion of the collagen-like domain from Gly45 to Pro80(domain 3). Wild type recombinant SP-A (SP-Ahyp; where hyp indicates hydroxyproline-deficient) and truncated mutant (TM) SP-As containing deletions of domain(s) 1 (TM1), 2 (TM2), 1 and 2 (TM1–2), and 1, 2, and 3 (TM1–2-3) were synthesized in insect cells and purified by mannose-Sepharose affinity chromatography. N-terminal disulfide-dependent dimerization was preserved at near wild type levels in the TM1–2 (at Cys−1) and TM2 proteins (at Cys−1 and Cys6), and to a lesser extent in TM1 (at Cys−1), but not in TM1–2-3. Cross-linking analyses demonstrated that the neck + CRD was sufficient for assembly of monomers into noncovalent trimers and that the N-terminal segment was required for the association of trimers to form higher oligomers. All TM proteins except TM1–2-3 bound to phospholipid, but only the N-terminal segment containing TM proteins aggregated phospholipid vesicles. The TM1, TM1–2, and TM2 but not the TM1–2-3 inhibited the secretion of surfactant from type II cells as effectively as SP-Ahyp, but the inhibitory activity of each mutant was blocked by excess α-methylmannoside and therefore nonspecific. TM1 and TM1–2-3 did not enhance the uptake of phospholipids by isolated type II cells, but the TM1–2 and TM2 had activities that were 72 and 83% of SP-Ahyp, respectively. We conclude the following for SP-A: 1) trimerization does not require the collagen-like region or interchain disulfide linkage; 2) the N-terminal portion of the collagen-like domain is required for specific inhibition of surfactant secretion but not for binding to liposomes or for enhanced uptake of phospholipids into type II cells; 3) N-terminal interchain disulfide linkage can functionally replace the N-terminal segment for lipid binding, receptor binding, and enhancement of lipid uptake; 4) the N-terminal segment is required for the association of trimeric subunits into higher oligomers, for phospholipid aggregation, and for specific inhibition of surfactant secretion and cannot be functionally replaced by disulfide linkage alone for these activities. Pulmonary surfactant lines the distal airspaces and stabilizes the gas exchanging alveolar units of the lung by reducing surface tension (1Goerke J. Clements J.A. Handbook of Physiology. 3. American Physiological Society, Bethesda1986: 247-261Google Scholar). Surfactant is composed of phospholipids and proteins that are organized as lattice-like arrays (e.g. tubular myelin) and vesicular aggregates within the alveolar lining fluid, and as a film at the alveolar air-liquid interface. Surfactant protein A (SP-A) 1The abbreviations used are: SP-A, surfactant protein A; TM, truncated mutant; CRD, carbohydrate recognition domain; IKC, isoleucine-lysine-cysteine; BSA, bovine serum albumin; DSG, disuccinimidyl glutarate; DPPC, dipalmitoylphosphatidylcholine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PAGE, polyacrylamide gel electrophoresis. is a hydrophilic, Ca2+-dependent phospholipid-binding protein that is secreted into the airspace by alveolar type II cells (2King R.J. Klass D.J. Gikas E.G. Clements J.A. Am. J. Physiol. 1973; 224: 788-795Crossref PubMed Scopus (193) Google Scholar). The function of SP-A is not fully understood, but reports that SP-A binds to a high affinity receptor on the surface of alveolar type II cells (3Wright J.R. Borchelt J.D. Hawgood S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5410-5414Crossref PubMed Scopus (102) Google Scholar, 4Kuroki Y. Mason R.J. Voelker D.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5566-5570Crossref PubMed Scopus (166) Google Scholar), is required for the formation and/or stability of tubular myelin (5Suzuki Y. Fujita Y. Kogishi K. Am. Rev. Respir. Dis. 1989; 140: 75-81Crossref PubMed Scopus (277) Google Scholar, 6Korfhagen T.R. Bruno M.D. Ross G.F. Huelsman K.M. Ikegami M. Jobe A.H. Wert S.E. Stripp B.R. Morris R.E. Glasser S.W. Bachurski C.J. Iwamoto H.S. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9594-9599Crossref PubMed Scopus (376) Google Scholar) and large surfactant aggregates (7Veldhuizen R.A. Yao L.J. Hearn S.A. J. 1996; PubMed Scopus Google Scholar), and surfactant surface activity in the of protein J. PubMed Scopus Google Scholar) or in surfactant The that SP-A is a protein is by in the of protein K. J. PubMed Google Sci. PubMed Scopus Google Scholar), reports that SP-A binds and Whitsett J.A. A. K. and Scholar), and the that the SP-A is to Bruno M.D. Huelsman K.M. Ross G.F. Whitsett J.A. T.R. J. Google Scholar). SP-A is a large composed of subunits that are by at or carbohydrate J.A. Ross J. PubMed Google Scholar, Mason R.J. Voelker D.R. J. PubMed Google Scholar) and in the N-terminal The by by in a containing of the of M. PubMed Scopus Google Scholar). The of the SP-A subunits are composed of domains including the following J. K. J. Hawgood S. PubMed Scopus Google Scholar, G.F. J. Whitsett J.A. J. PubMed Google 1) a N-terminal 2) a collagen-like of is and is or containing a 3) a neck and 4) a carbohydrate recognition domain association of subunits by the of the collagen-like domains into R.J. 1989; PubMed Scopus Google Scholar). The of the neck domain in the assembly of SP-A not but in the formation of in region is for trimerization PubMed Scopus Google Scholar). SP-A is a of trimers that are the N-terminal segment and the of the collagen-like domain and by and interchain disulfide at Cys−1 and R.J. 1989; PubMed Scopus Google Scholar, J. J. 1988; PubMed Scopus Google Scholar). The domains of SP-A that type II cells and surfactant phospholipids by The CRD of the binding for the surface of the the high affinity SP-A receptor on type II cells, and surfactant Voelker D.R. M.D. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar, Y. Y. Mason R.J. Voelker D.R. J. PubMed Google Scholar, Y. Mason R.J. Voelker D.R. J. PubMed Google Scholar). We that the N-terminal domains that to including the interchain disulfide at and the collagen-like are for SP-A function S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). The collagen-like region was required for binding to the SP-A receptor and the specific inhibition of surfactant secretion from type II of the interchain disulfide by of for blocked the of liposomes by SP-A and the inhibitory of SP-A on surfactant secretion from isolated alveolar type II analyses that the N-terminal and the interchain at is not for the of SP-A type II or surfactant phospholipids M. M. PubMed Scopus Google Scholar). the current used truncated mutant of SP-A to examine the of the N-terminal segment and of the collagen-like domain in phospholipid binding and aggregation, and SP-A The of the mutant recombinant proteins TM1, and TM1–2-3 M. PubMed Scopus Google Scholar). The TM2 protein was by the TM2 mutant the of of SP-A was by of a SP-A 1989; PubMed Scopus Google Scholar, K. J. Mason R.J. Y. J. Voelker PubMed Scopus Google Scholar). The TM2 was into the of and the was of the region of the TM2 the and the of S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). A recombinant containing the TM2 for SP-A was by in cells following and the of cells were recombinant at a of of and and for 72 SP-A was purified from the by to mannose-Sepharose in the of 1 and 2 J. PubMed Scopus Google Scholar). The purified recombinant SP-A was and at Surfactant was isolated by of J. PubMed Scopus Google Scholar) and on S. R.J. PubMed Scopus Google Scholar, Y. Mason R.J. Voelker D.R. J. 1988; PubMed Google Scholar). SP-A was isolated and purified from the surfactant by mannose-Sepharose affinity J. PubMed Scopus Google Scholar), and gel The SP-A of containing recombinant SP-A was a SP-A a A. Voelker D.R. PubMed Scopus Google Scholar). The of of the was and the from to protein were the protein bovine serum as a The of on the of the recombinant proteins were by SP-As were the disuccinimidyl in containing and at for The was by of reducing and the proteins were on and to the of N-terminal deletions on the inhibition of surfactant secretion were as Mason R.J. Voelker D.R. J. PubMed Google Scholar). alveolar type II cells were isolated from by and on Mason R.J. J. PubMed Scopus Google Scholar). The type II cells were into and in containing and serum at in a SP-A were for to surfactant secretion by the for 3 was the and SP-A to the SP-A was excess was as a for surfactant and as secretion in the in the cells + A binding was to the of recombinant of SP-A to SP-A for receptor on the surface of isolated type II cells Y. Mason R.J. Voelker D.R. J. 1988; PubMed Google Scholar). 2 type II were in at in a The following the cells were by the at of containing 1 The were 1 SP-A and recombinant SP-As in for 3 at in a on containing 2 and 1 BSA, the cells were in and was in a binding and were from as Mason R.J. Voelker D.R. J. PubMed Google Scholar). were by of lipid composed of and SP-As by in at was by at the of lipid binding, liposomes by of the lipid were SP-A in A containing and for 1 at the was at for and and the SP-A of the and were by binding was as + in liposomes or were were of phospholipid liposomes by type II cells was to the of J.R. R.E. Hawgood S. Clements J.A. J. PubMed Google Scholar) Y. Mason R.J. Voelker D.R. J. PubMed Google Scholar). isolated alveolar type II cells were liposomes composed of and SP-A in of for 1 at The and cells were by at for at and the cells were in containing 1 of of was to each and the cells and were to and uptake was to the The of the mutant recombinant proteins used in study are in SP-A that is in insect cells functional that are to the of in the collagen-like region for hydroxyproline-deficient) Mason R.J. Voelker D.R. J. PubMed Google Scholar). the of study the domains of SP-A from to as the domain 1 domain 2 of the collagen-like domain 3 of the collagen-like and The and of the mutant recombinant SP-As containing deletions from Asn1 the of the N-terminal segment or the of the collagen-like region or and of the collagen region or M. PubMed Scopus Google Scholar). mutant SP-A containing a of the collagen-like region or was The TM2 the on reducing the of the SP-Ahyp, the TM2 at as large as is interchain disulfide formation at are for interchain at Cys−1 and We that disulfide formation at Cys−1 in TM1 and TM1–2 in and on of SP-Ahyp, by is and higher M. PubMed Scopus Google Scholar). The TM1–2-3 is to the of the at the of the to at the the that in SP-Ahyp, TM1, and TM1–2 M. PubMed Scopus Google Scholar). The of and near wild type levels of disulfide at the of TM1 and TM1–2, for of the functional of the deletions that are from that are to of interchain that to the assembly of the SP-A were by and the are in 3. of the by on reducing in the of a of that the of were in the as gel are assembly of S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). Cross-linking analyses that the TM1–2-3 was a noncovalent that collagen-like region and interchain disulfide formation are not required for of SP-A or to the association of in the fully The TM1 and TM1–2 did not form but the TM2 was at that N-terminal interchain disulfide linkage is not sufficient for the association of trimers and that the N-terminal segment or interchain are required for the We that of or Cys−1 by did not the assembly of large SP-A oligomers, on analyses and gel S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar, M. M. PubMed Scopus Google Scholar). these that the neck + CRD are sufficient for trimerization of SP-A, and that the N-terminal segment in the association of trimeric subunits in that is not to disulfide of TM2 SP-A, wild type recombinant SP-A and TM2 SP-As were to reducing and and of association of of TM The wild type recombinant SP-A TM1, TM1–2, or TM2 were the for at and on reducing and The of N-terminal deletions on the binding of TM SP-As to liposomes was by the of protein that the phospholipid as Mason R.J. Voelker D.R. J. PubMed Google Scholar). binding was by of SP-A in the of from Ca2+-dependent in of bound to the of TM1 and of The TM2 protein wild type levels of phospholipid binding activity but binding by TM1–2-3 was We that the binding of to lipid is that of S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). that domain 2 of SP-A does not to lipid binding and that domain 3 is required for wild type levels of lipid binding The that TM1–2 bound to lipid as effectively as that N-terminal disulfide linkage can functionally replace the N-terminal segment to lipid We that the interchain disulfide but not the collagen-like domain of SP-A is required for of phospholipid liposomes S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). The of the N-terminal segment in was by of truncated mutant of SP-A liposomes in the of and of in the for by was The TM1 only for of A The TM1–2 and TM1–2-3 were in the of and respectively. of the of function in TM1–2-3 and the wild type activity of the as S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar) indicates that interchain or the N-terminal segment or are required for S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar), but not N-terminal interchain are or of Cys−1 that interchain does not M. M. PubMed Scopus Google Scholar). The activity of TM2 was to SP-Ahyp, a of of the TM1–2 and TM2 that the N-terminal and not N-terminal interchain in near wild type levels in is required for lipid by these are a for SP-A the N-terminal segment domain in the of phospholipid vesicles. The binding of mutant SP-As to the receptor on the surface of isolated type II cells was in a binding and the are in SP-A for receptor in a of to Mason R.J. Voelker D.R. J. PubMed Google Scholar, Voelker D.R. M.D. PubMed Scopus Google Scholar, Y. Mason R.J. Voelker D.R. J. PubMed Google Scholar). The TM1 and interchain linkage at the did not for receptor The TM1–2 and the TM2 SP-A for receptor binding that were and that N-terminal interchain disulfide formation is for the activity of SP-A to for receptor and that domain 2 and the N-terminal segment are not of the preserved activity of TM2 and the of binding by the protein S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar) that domain 3 in the binding of SP-A to the We that of the interchain disulfide inhibition of surfactant secretion by SP-A, but of the collagen-like domain only specific inhibition S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). the the N-terminal segment and of the collagen domain in inhibition of secretion by SP-A The of required for inhibition of secretion was was higher of Mason R.J. Voelker D.R. J. PubMed Google Scholar, Voelker D.R. M.D. PubMed Scopus Google Scholar, S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar, Y. Mason R.J. Voelker D.R. J. PubMed Google Scholar). by TM1, TM1–2, and TM2 was that SP-Ahyp, of and respectively. to the inhibition by these proteins and the protein S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar), the TM1–2-3 was a of surfactant secretion indicates that interchain disulfide linkage is for inhibition of surfactant the of inhibition by the TM the was in the of a that to the inhibition of surfactant secretion by as A Y. Mason R.J. Voelker D.R. J. 1988; PubMed Google Scholar). The inhibition by of the TM proteins was at blocked by excess is for SP-A or these that interchain disulfide formation at Cys−1 can functionally replace the N-terminal segment and/or the disulfide to inhibition of surfactant specific inhibition of surfactant secretion the N-terminal segment and the N-terminal and C-terminal of the collagen-like of of inhibition of secretion by TM of secretion by SP-Ahyp, TM1, TM1–2, or was as in except that was to the We that of the collagen-like domain and of the disulfide blocked the uptake of lipid by type II cells S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). the of SP-A, of the was by the cells the of SP-Ahyp, was a a in lipid to The truncated proteins the of N-terminal disulfide TM1–2 and enhanced uptake by to and to respectively. The TM1, is only had a on lipid uptake enhancement to and the is to disulfide was enhancement to We conclude that interchain disulfide linkage but not domain 1 or domain 2 is for lipid uptake by type II The that TM2 is as as and that is in the S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar) that the within domain 3 a in lipid uptake by The of study was to examine the functional of the N-terminal domains of SP-A including the interchain disulfide the N-terminal and the N-terminal and C-terminal of the collagen-like The was to N-terminal deletions the and of the collagen-like region of SP-A and the of the on the of SP-A type II cells and surfactant of TM proteins that the N-terminal segment and SP-Ahyp, TM1–2 and and TM1–2-3 and of the function of each of the N-terminal of N-terminal disulfide at Cys−1 in TM1 and TM1–2 of the functional of of the N-terminal segment and the N-terminal of the collagen-like domain the of of interchain from study and reports that the domains of SP-A that are or not for SP-A are in of SP-A that are or for assembly from monomers used to assembly binding binding inhibition of secretion inhibition of secretion uptake used to in a The assembly of SP-A can be in the of subunits into trimers and the association of trimers into domains of SP-A in the trimerization of SP-A as the neck the collagen-like and interchain disulfide The that SP-A as a noncovalent indicates that in the neck + CRD are sufficient to the association of J. K. J. Hawgood S. Clements J.A. Am. J. Physiol. 1989; Scholar). analyses of that the and that the neck form of PubMed Scopus Google Scholar, K. PubMed Scopus Google K. PubMed Scopus Google Scholar). from study are that Cross-linking analyses that the TM1–2-3 was composed of that the neck domain is sufficient for trimeric indicates that of the neck + CRD into a functional by to does not require the collagen-like the N-terminal or interchain disulfide and that the of SP-A from the to the as in and K. J. 1996; PubMed Scopus Google Scholar). the of trimeric subunits the N-terminal domains including the collagen-like the N-terminal and the N-terminal interchain disulfide We mutant proteins containing of the interchain disulfide the Cys−1 interchain disulfide or of the collagen-like domain S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar, M. M. PubMed Scopus Google Scholar). interchain disulfide or the collagen-like region were to be required for assembly of SP-A into a the of the wild type recombinant protein S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). the current the assembly of SP-A was by The of TM1–2 that was by the analyses was a that the C-terminal of the collagen-like domain and disulfide at the were not sufficient to the association of trimeric subunits into higher oligomers. The mutant protein TM2 was a the N-terminal segment in the trimeric subunits of of the N-terminal segment does not to be to the of interchain disulfide and gel that the SP-Ahyp, and SP-Ahyp, were composed of trimeric subunits S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar, M. M. PubMed Scopus Google Scholar). the that and SP-A Y. Mason R.J. Voelker D.R. J. 1988; PubMed Google Scholar), SP-Ahyp, S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar), and SP-Ahyp, M. M. PubMed Scopus Google Scholar) as high are the that noncovalent in the collagen-like and the N-terminal and not interchain disulfide are the of SP-A assembly into trimeric and oligomers. SP-A binds to surfactant and to phospholipid liposomes that binding can be blocked by in the CRD, that the in that domain Voelker D.R. M.D. PubMed Scopus Google Scholar). in the N-terminal region binding to lipid vesicles. We that the of the interchain disulfide binding to liposomes by S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). of the N-terminal segment lipid binding by only of lipid binding by TM1 to dimerization by disulfide at Cys−1 in TM1 the SP-Ahyp, binding by the TM1–2 is was of that of SP-Ahyp, and wild type levels of lipid binding were by the N-terminal of the collagen-like domain as for lipid binding and that N-terminal interchain disulfide linkage can functionally replace the N-terminal segment to lipid interchain disulfide are not required for lipid binding of Cys−1 was M. M. PubMed Scopus Google Scholar), but the lipid binding activity of TM2 TM1–2 that of the N-terminal segment carbohydrate to lipid The of the TM proteins the of the C-terminal of the collagen-like region in lipid The only the neck + CRD of SP-A and is not did not lipid binding in but the N-terminal segment containing had lipid binding activity that was that of S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). of lipid binding activities of TM2 and indicates that the C-terminal of the collagen-like domain to lipid binding S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). is blocked by of the disulfide S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar) and by in the CRD Voelker D.R. M.D. PubMed Scopus Google Scholar). of the N-terminal segment lipid aggregation, and the N-terminal deletions in TM1–2 and TM1–2-3 the of the N-terminal segment to the the TM1–2-3 protein the S. J. K. Voelker D.R. J. PubMed Scopus Google that interchain disulfide the N-terminal segment and/or the N-terminal are required for the of the TM1–2 and TM2 proteins the of these N-terminal domains in lipid the lipid binding function of SP-A, disulfide formation at Cys−1 in the TM1–2 mutant not functionally replace the N-terminal segment in the the N-terminal segment containing TM2 aggregated liposomes as as The of a interchain (at within the N-terminal is not a for in function TM1–2 and that only interchain disulfide is sufficient M. M. PubMed Scopus Google Scholar). for that is by a the CRD and carbohydrate J. PubMed Scopus Google Scholar, Hawgood S. K. J. PubMed Google Scholar), is to Asn1 of the TM2 but is from the of the We that is on that recombinant SP-A by in the of or of the Asn1 and for aggregates lipid Mason R.J. Voelker D.R. J. PubMed Google Scholar). these that the N-terminal segment is required for and that the N-terminal interchain disulfide is required but is not sufficient for the We a for in SP-A binds to phospholipid the CRD and liposomes by N-terminal binding to the SP-A receptor and is not to the of excess Y. Mason R.J. Voelker D.R. J. 1988; PubMed Google Scholar). that SP-A receptor binding the receptor recognition in the CRD and domains in including the collagen-like domain and interchain disulfide in the CRD Voelker D.R. M.D. PubMed Scopus Google Scholar, Y. Mason R.J. Voelker D.R. J. PubMed Google Scholar) and of the collagen-like domain S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar) binding to the SP-A of the TM1–2 and TM2 proteins was used to domains within the collagen-like region that are required for receptor for receptor by the TM2 protein was at SP-A but was to at the The TM1–2 is for binding, at a that was or The TM1 did not for receptor of the of interchain disulfide linkage at the of these is that the N-terminal segment is not for receptor binding and can be functionally replaced by interchain disulfide linkage at the The C-terminal of the collagen-like domain to receptor binding, the TM1–2 and TM2 proteins were and the protein was to the SP-A receptor on type II cells is to inhibition of secretion of can surfactant activities are blocked by excess carbohydrate and are therefore Y. Mason R.J. Voelker D.R. J. 1988; PubMed Google Scholar). inhibition of secretion by SP-A is to the of We that of the collagen-like domain of SP-A specific inhibition of surfactant secretion S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). The TM1 in indicates in to the collagen-like the N-terminal segment is required for specific inhibition of surfactant inhibition by the TM2 protein that at of specific inhibition within the collagen-like region to the N-terminal of the The of the CRD is for specific a mutant protein containing the to and to inhibition Y. Mason R.J. Voelker D.R. J. PubMed Google Scholar). these that the and carbohydrate binding region of the CRD, the N-terminal and the collagen-like domain are for specific inhibition of surfactant secretion by SP-A be required to within the C-terminal of the collagen-like domain and the neck region to specific inhibition of surfactant The that the TM1 and TM1–2 were of surfactant secretion indicates that the N-terminal segment is not required for of disulfide is the is of surfactant the TM1–2-3 is a The that the for inhibition of secretion by SP-A the neck + CRD and at the The of study and that uptake of into isolated type II cells the collagen-like interchain disulfide and the the activity of the TM in the uptake the extent of disulfide linkage at the The TM1–2 and TM2 proteins near wild type levels of interchain linkage at the enhanced lipid uptake to extent that was to the the TM1 protein is had activity in the The interchain mutant SP-Ahyp, was only a of lipid uptake S. J. K. Voelker D.R. J. PubMed Scopus Google Scholar). of the preserved functional activity of TM2 and the activity of the that the but not the portion of the collagen-like domain of SP-A that are for the of SP-As to enhance the association of into type II of the N-terminal domains of SP-A that lipid binding interchain disulfide lipid the N-terminal segment and interchain receptor and uptake of surfactant by type II cells N-terminal interchain and the C-terminal of the collagen-like and specific inhibition of surfactant secretion N-terminal domains except the of the the SP-A for the formation of tubular for the of surfactant to protein and for