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The gene coding for the β-chain of C4b-binding protein (C4BPB) has become a pseudogene in the mousE
47 COMPLEMENT IS PACKAGED
RECEPTOR TYPE 1 (CR1, CD35) IN SECONDARY GRANULES OF
HUMAN NEUTROPHILS
(PMN).
John M. Robinson* and Rick Jack# *Department of Cell Biolog3~/Neurobiology/Anatomy, Ohio State University, Columbus, OH; #Department of Medicine, Harvard Medical School, Boston, MA. USA. Replicate samples of human PMN purified at 4°C were incubated at 4°C or at 37oc with 10-7M fMLP. PMN were disrupted by cavitation and fraetionated on a 30-00% sucrose gradient. Gradients were analyzed by standard enzymatic criteria and pooled into plasma membrane (PM), secondary (2 °) granule and primary (1 o) granule fractions. The pools were radioiodinated and immunoprecipitated with mAb specific for CR1 and CR3, resolved by SDS-PAGE and analyzed by autoradiography. In unperturbed 4 o c PMN, the majority of labeled CRI was colocallzed with CR3 in the lactoferrin positive 2 ° granule fraction. The remainder of the labeled CR 1 and CR3 were associated with the PM. No labeled CR1, CR3 or MHC class I was found in the 1° granule compartment. In the fMLP-treated PMN, translocation of CR1 and CR3 to the PM was increased compared to 4oc controls while the amount of receptors in the 2 ° granule compartment decreased. Intracellular localization of CR1 to the 2 ° granule compartment was confirmed by confocal microscopy. PMN (purified at 4°C and held at 4°C or activated with fMLP) were stained for intracellular and plasma membrane expression of CRI, CR3 and lactoferrin and examined. In 4°C PMN, CRI was found to localize in a granule compartment morphologically similar to that which contained lactoferrin in 2 ° granules. When PMN were activated, the majority of CR1 was found on the cell surface and not intracellularly. Thus, CRI colocalizes with lactoferrin and CR3 and appears to be a component of secondary granules of unperturbed PMN.
T H E G E N E C O D I N G F O R T H E 13-CHAIN O F C4bBINDING PROTEIN (C4BPB) H A S B E C O M E A PSEUDOGENE IN THE MOUSE.
S. Rodriguez de C6rdoba, M. Perez-Blas, R. Ramos-Rniz P. S/mchez-Corral, F, Pardo-Manuel de Villena and J. Rey-Campos. Unidad de Inmunologia, Centro de Investigaeiones Biolbgicas (CSIC), Vel~izquez 144, 28006Madrid, SPAIN. CABPJ3 is one of the two polypeptides that in humans organize the plasma glycoprotein C4b-binding protein (C4BP). C4BPI3 binds the anticoagulant vitamin K-dependent protein S. Two, non-mutually exclusive, roles have been proposed for the C4BP-pretein S interaction. It has been suggested that it plays a role in the control of the protein C anticoagulatory pathway. In addition, it may serve an important role in localizing C4BP to the surface of injured or activated cells. While the physiological significance of CABP-protein S interaction is unclear, it has clinical relevance because elevated plasma levels of C4BP are associated with increased risk to thromboembolic disorders in humans, due to an inactivation of the protein C anticoagulatory pathway. Using a human CABP~i--cDNA probe we have isolated and characterized a genomic DNA fragment that includes the routine C4BPB gone. Murine C4BPB is a single copy gene that maps close to the C4BPA gene in chromosome 1. It contains two exons homologous to the exons coding for the SCR-I and SCR-2 repeats of the human C4BP~I polypeptide chain. Sequence analysis of the C4BPB-exons in the Mus musculus inbred strains CBA, Balb/c, C57BL/6 and in pen-bred Swiss mice, and in Mus spretus demonstrated the presence of two in-phase stop codans which are incompatible with the expression of a functional C4BP[~ polypeptide. The loss of a functional C4BPB gone has been a relatively recent event in the evolution of the mouse. The characterization of the murine C4BPBgone documents the peculiar situation of a single copy gene that is fimctional in humans,but has become a pseudogene in the mouse.
E F F E C T S O F H U M A N CD59 IN AN IN V I T R O MODEL FOR HYPERACUTE REJECTION, Catherine A. Rogers, Carmen W. van den Berg and B.Paul Morgan Dept.Medical Biochemistry,University of Wales College of Medicine,Cardiff, U.K. Xenotransplantation, specifically the use of pig organs in human recipients, has become an important aim in transplant immunology. The usual fate of xenografls is complement-mediated hyperacute rejection and suggested strategies for subverting this process include the incorporation or expression of human complement regulatory molecules in the donor organ. We have recently purified the pig analogue of human CD59 and shown it to be species non-restricted. This finding raises the possibility that expression of human CD59 on pig organs will not offer a significant survival advantage. In order to further explore this possibility we have utiliscd porcine endothelial cells (PECs) as an in vitro model. Incorporation of human CD59 into PECs, assessed by flow cytometry, was extremely inefficient when compared with incorporation into several human cell lines. Even at very high offered doses of CD59 only a small amount of incorporation was obtained and killing by human serum was not inhibited. These results are in contrast to those obtained with human cell lines such as K562 where CD59 incorporated efficiently and inhibited killing in a dose-dependent manner, and to published studies in which DAF was shown to reincorporate efficiently and protect PECs. The failure to incorporate CD59 into PECs prevents us from assessing the likely efficacy of expressed CD59 and eliminates the possibility of infusion as a means of delivering CD59 to the endothelia of porcine organs.
CARRIAGE OF COMPLEMENT REGULATORY PROTEINS BY VESICLES (PROSTASOMES) IN SEMINAL PLASM~ Rooney IA and A t k i n s o n JP Dept of Medicine, W a s h i n g t o n U n i v e r s i t y school of Medicine, St Louis. We p r e v i o u s l y d e m o n s t r a t e d that cells, including spermatozoa, acquire f u n c t i o n i n g m o l e c u l e s of CD59, a g l y c o s y l p h o s p h a t i d y l i n o s i t o l (GPI)-linked c o m p l e m e n t regulatory protein, d u r i n g interaction with CD59-bearing vesicles (prostasomes) in seminal plasma (SP) (d Exp Med; 177:1409). We now show that two other G P I - l i n k e d p r o t e i n s are carried by these particles. We i d e n t i f i e d d e c a y a c c e l e r a t i n g factor (DAF; CD55) in SP at a c o n c e n t r a t i o n of 500 ng/ml, 30% of w h i c h was p r o s t a s o m e - a s s o c i a t e d . On W e s t e r n blots p r o s t a s o m e DAF a p p e a r e d as two bands of 65 and 70 kDa. We also found that p r o s t a s o m e s c a r r y CDw52, a OPI-linked, l y m p h o c y t e antigen of u n c e r t a i n function. P r o s t a s o m e CDw52 was c o m p a r a b l e in size (25-29 kDa) w i t h CDw52 of other origins. O t h e r s h a v e shown t~at CDw52 in SP has the ability to b i n d to cells (J Reprod Immunol; 23:189) a l t h o u g h the m e c h a n i s m w h i c h permits this p h e n o m e n o n was unknown. Our data show that at least 90% of SP cnw52 is p r o s t a s o m e bound and suggest that cells a c q u i r e this protein, like CD59, d i r e c t l y from prostasomes. Both DAF and CDw52 w e r e i m m u n o p r e c i p i t a t e d from SP u s i n g anti-CD59 a n t i b o d y coupled to s o l i d phase, d e m o n s t r a t i n g that all three p r o t e i n s were present on the same particles. In contrast, w e failed to detect the t r a n s m e m b r a n e c o m p l e m e n t inhibitor membrane c o f a c t o r p r o t e i n (MCP; CD46) on prostasomes: they may t h e r e f o r e carry G P I - l i n k e d p r o t e i n s selectively. T h e s e d a t a strengthen our h y p o t h e s i s that p r o s t a s o m e s p r o v i d e a r e s e r v o i r of G P I - l i n k e d proteins from w h i c h p r o t e i n s lost from s p e r m a t o z o a can be replenished, and suggest a b i o l o g i c role for a GPI anchor.
RECEPTOR TYPE 1 (CR1, CD35) IN SECONDARY GRANULES OF
HUMAN NEUTROPHILS
(PMN).
John M. Robinson* and Rick Jack# *Department of Cell Biolog3~/Neurobiology/Anatomy, Ohio State University, Columbus, OH; #Department of Medicine, Harvard Medical School, Boston, MA. USA. Replicate samples of human PMN purified at 4°C were incubated at 4°C or at 37oc with 10-7M fMLP. PMN were disrupted by cavitation and fraetionated on a 30-00% sucrose gradient. Gradients were analyzed by standard enzymatic criteria and pooled into plasma membrane (PM), secondary (2 °) granule and primary (1 o) granule fractions. The pools were radioiodinated and immunoprecipitated with mAb specific for CR1 and CR3, resolved by SDS-PAGE and analyzed by autoradiography. In unperturbed 4 o c PMN, the majority of labeled CRI was colocallzed with CR3 in the lactoferrin positive 2 ° granule fraction. The remainder of the labeled CR 1 and CR3 were associated with the PM. No labeled CR1, CR3 or MHC class I was found in the 1° granule compartment. In the fMLP-treated PMN, translocation of CR1 and CR3 to the PM was increased compared to 4oc controls while the amount of receptors in the 2 ° granule compartment decreased. Intracellular localization of CR1 to the 2 ° granule compartment was confirmed by confocal microscopy. PMN (purified at 4°C and held at 4°C or activated with fMLP) were stained for intracellular and plasma membrane expression of CRI, CR3 and lactoferrin and examined. In 4°C PMN, CRI was found to localize in a granule compartment morphologically similar to that which contained lactoferrin in 2 ° granules. When PMN were activated, the majority of CR1 was found on the cell surface and not intracellularly. Thus, CRI colocalizes with lactoferrin and CR3 and appears to be a component of secondary granules of unperturbed PMN.
T H E G E N E C O D I N G F O R T H E 13-CHAIN O F C4bBINDING PROTEIN (C4BPB) H A S B E C O M E A PSEUDOGENE IN THE MOUSE.
S. Rodriguez de C6rdoba, M. Perez-Blas, R. Ramos-Rniz P. S/mchez-Corral, F, Pardo-Manuel de Villena and J. Rey-Campos. Unidad de Inmunologia, Centro de Investigaeiones Biolbgicas (CSIC), Vel~izquez 144, 28006Madrid, SPAIN. CABPJ3 is one of the two polypeptides that in humans organize the plasma glycoprotein C4b-binding protein (C4BP). C4BPI3 binds the anticoagulant vitamin K-dependent protein S. Two, non-mutually exclusive, roles have been proposed for the C4BP-pretein S interaction. It has been suggested that it plays a role in the control of the protein C anticoagulatory pathway. In addition, it may serve an important role in localizing C4BP to the surface of injured or activated cells. While the physiological significance of CABP-protein S interaction is unclear, it has clinical relevance because elevated plasma levels of C4BP are associated with increased risk to thromboembolic disorders in humans, due to an inactivation of the protein C anticoagulatory pathway. Using a human CABP~i--cDNA probe we have isolated and characterized a genomic DNA fragment that includes the routine C4BPB gone. Murine C4BPB is a single copy gene that maps close to the C4BPA gene in chromosome 1. It contains two exons homologous to the exons coding for the SCR-I and SCR-2 repeats of the human C4BP~I polypeptide chain. Sequence analysis of the C4BPB-exons in the Mus musculus inbred strains CBA, Balb/c, C57BL/6 and in pen-bred Swiss mice, and in Mus spretus demonstrated the presence of two in-phase stop codans which are incompatible with the expression of a functional C4BP[~ polypeptide. The loss of a functional C4BPB gone has been a relatively recent event in the evolution of the mouse. The characterization of the murine C4BPBgone documents the peculiar situation of a single copy gene that is fimctional in humans,but has become a pseudogene in the mouse.
E F F E C T S O F H U M A N CD59 IN AN IN V I T R O MODEL FOR HYPERACUTE REJECTION, Catherine A. Rogers, Carmen W. van den Berg and B.Paul Morgan Dept.Medical Biochemistry,University of Wales College of Medicine,Cardiff, U.K. Xenotransplantation, specifically the use of pig organs in human recipients, has become an important aim in transplant immunology. The usual fate of xenografls is complement-mediated hyperacute rejection and suggested strategies for subverting this process include the incorporation or expression of human complement regulatory molecules in the donor organ. We have recently purified the pig analogue of human CD59 and shown it to be species non-restricted. This finding raises the possibility that expression of human CD59 on pig organs will not offer a significant survival advantage. In order to further explore this possibility we have utiliscd porcine endothelial cells (PECs) as an in vitro model. Incorporation of human CD59 into PECs, assessed by flow cytometry, was extremely inefficient when compared with incorporation into several human cell lines. Even at very high offered doses of CD59 only a small amount of incorporation was obtained and killing by human serum was not inhibited. These results are in contrast to those obtained with human cell lines such as K562 where CD59 incorporated efficiently and inhibited killing in a dose-dependent manner, and to published studies in which DAF was shown to reincorporate efficiently and protect PECs. The failure to incorporate CD59 into PECs prevents us from assessing the likely efficacy of expressed CD59 and eliminates the possibility of infusion as a means of delivering CD59 to the endothelia of porcine organs.
CARRIAGE OF COMPLEMENT REGULATORY PROTEINS BY VESICLES (PROSTASOMES) IN SEMINAL PLASM~ Rooney IA and A t k i n s o n JP Dept of Medicine, W a s h i n g t o n U n i v e r s i t y school of Medicine, St Louis. We p r e v i o u s l y d e m o n s t r a t e d that cells, including spermatozoa, acquire f u n c t i o n i n g m o l e c u l e s of CD59, a g l y c o s y l p h o s p h a t i d y l i n o s i t o l (GPI)-linked c o m p l e m e n t regulatory protein, d u r i n g interaction with CD59-bearing vesicles (prostasomes) in seminal plasma (SP) (d Exp Med; 177:1409). We now show that two other G P I - l i n k e d p r o t e i n s are carried by these particles. We i d e n t i f i e d d e c a y a c c e l e r a t i n g factor (DAF; CD55) in SP at a c o n c e n t r a t i o n of 500 ng/ml, 30% of w h i c h was p r o s t a s o m e - a s s o c i a t e d . On W e s t e r n blots p r o s t a s o m e DAF a p p e a r e d as two bands of 65 and 70 kDa. We also found that p r o s t a s o m e s c a r r y CDw52, a OPI-linked, l y m p h o c y t e antigen of u n c e r t a i n function. P r o s t a s o m e CDw52 was c o m p a r a b l e in size (25-29 kDa) w i t h CDw52 of other origins. O t h e r s h a v e shown t~at CDw52 in SP has the ability to b i n d to cells (J Reprod Immunol; 23:189) a l t h o u g h the m e c h a n i s m w h i c h permits this p h e n o m e n o n was unknown. Our data show that at least 90% of SP cnw52 is p r o s t a s o m e bound and suggest that cells a c q u i r e this protein, like CD59, d i r e c t l y from prostasomes. Both DAF and CDw52 w e r e i m m u n o p r e c i p i t a t e d from SP u s i n g anti-CD59 a n t i b o d y coupled to s o l i d phase, d e m o n s t r a t i n g that all three p r o t e i n s were present on the same particles. In contrast, w e failed to detect the t r a n s m e m b r a n e c o m p l e m e n t inhibitor membrane c o f a c t o r p r o t e i n (MCP; CD46) on prostasomes: they may t h e r e f o r e carry G P I - l i n k e d p r o t e i n s selectively. T h e s e d a t a strengthen our h y p o t h e s i s that p r o s t a s o m e s p r o v i d e a r e s e r v o i r of G P I - l i n k e d proteins from w h i c h p r o t e i n s lost from s p e r m a t o z o a can be replenished, and suggest a b i o l o g i c role for a GPI anchor.