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Secretion of human latent TGF-ß-binding protein-3 (LTBP-3) is dependent on co-expression of TGF-ß

Carita Penttinen, Juha Saharinen, Krista Weikkolainen, Marko Hyytiäinen and Jorma Keski-Oja*

Departments of Virology and Pathology, Haartman Institute and Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland



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  		Fig. 2. Structure and relatedness of LTBPs. (A) Schematic illustration of the structures of human LTBP 1-4 and an alternative hL3+EGF splice variant. hL3+EGF contains an additional Ca2+-binding EGF-like repeat between the 13th and the 14th repeat units. The protein domains, which were included in phL3/911-1153 cDNA construct used in recombinant protein production for immunizations, are also illustrated. (B) A dendrogram showing the relationships and similarity percentages between the protein sequences of human LTBP 1-4 and mouse Ltbp-3. The amino acid sequences were retrieved from GenBank and aligned with ClustalX. The alignment is displayed by TREEVIEW.

 


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  		Fig. 1. Cloning and sequencing of human LTBP-3. (A) A schematic presentation of the obtained cDNA and of some of the recognition sites of the restriction enzymes, which were used in the process of cloning. (B) The nucleotide and deduced amino-acid sequence of human LTBP-3. Nucleotide numbering starts from the proposed upstream translation initiation codon. Exon-intron boundaries are marked with vertical lines and EGF-like repeats are shaded with gray. 8-Cys repeats and the hybrid domains are indicated by patterns of eight circled cysteines. An alternative splice site of hL3+EGF is marked with an asterisk (*). Specified arrows are used to show the 5' coding ends of phL3 and phL3+ATG cDNA expression constructs.

 


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  		Fig. 3. Analyses of the mRNA expression levels of hLTBP-3 and hL3+EGF splice variant in various human tissues and cell lines. (A) Human multi tissue northern blot was hybridized with a radioactively labeled cDNA fragment of hLTBP-3. A single 4.6 kb mRNA species corresponding to human LTBP-3 was detected. Hybridization to human ß-actin was used to control the loading of RNA. (B) Total RNA was extracted from various human cell lines, separated on a formaldehyde-agarose gel and immobilized on a nylon filter. Northern hybridization analysis was carried out using the hLTBP-3 cDNA probe. The lower panel shows the hybridization signals of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), which were used to control the RNA loading. (C) RT-PCR and Southern hybridization analyses of cDNA prepared from different human tissues using primers specific for hL3+EGF splice variant. The expected amplification product migrated at 154 bp. The lower panel illustrates RT-PCR amplification of an abundantly expressed G3PDH in the corresponding tissues.

 


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  		Fig. 4. hLTBP-3 protein is expressed in transfected COS-7 cells, and its secretion is affected by co-expression of TGF-ß1. (A) COS-7 cells were plated on glass coverslips and transfected with the full-length expression construct of hLTBP-3. The cells were fixed three days after transfection and subjected to immunofluorescence analysis using the affinity-purified hLTBP-3 antibody (Ab-hL3/2925). White arrows and outlining are used to mark the two cells showing an intense fluorescent signal of the translation product of hLTBP-3. (B) COS-7 cells were transfected with the cDNA expression constructs indicated on the figure. Conditioned cell culture medium was collected 3 days after transfection and analyzed by immunoblotting. The proteins were separated by 7.5% SDS-PAGE under non-reducing conditions and subjected to immunoblot analysis using an anti-hLTBP-3 (Ab-hL3/2925) antibody. An arrow indicates the 240 kDa complexes, which are secreted when either of the indicated two full-length hLTBP-3 cDNAs and pTGF-ß1 are co-transfected. An arrowhead on the left indicates the smaller complex, which is secreted following the co-transfection of phL3/699-1153 expression construct and pTGF-ß1. The same symbols are used in C. (C) Similar complexes recognized by an antibody specific to TGF-ß1•LAP. Immunoblotting was carried out as defined above with the exception that anti-ß1•LAP antibody (680) was used in immunodetection. Free ß1•LAP and unprocessed TGF-ß1•LAP dimers are marked by a bracket. (D) Immunoprecipitation analysis of the conditioned medium of transiently transfected COS-7 cells using anti-hLTBP-3 (Ab-hL3/2925) antibody. The samples were separated by SDS-PAGE using 4-15% gradient gels under reducing conditions.

 


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  		Fig. 5. hLTBP-3 is secreted by osteosarcoma cells as high molecular weight complexes. (A) Concentrated cell culture medium of MG-63, U-2OS and Saos-2 osteosarcoma cells was collected after culturing for 3 days under serum-free conditions. The protein samples were separated by 7.5% SDS-PAGE under non-reducing conditions and blotted onto nitrocellulose filters. Conditioned mediums from untransfected COS-7 cells and COS-7 cells overexpressing hLTBP-3 and TGF-ß1 were used as controls. Immunodetection was carried out using the affinity-purified anti-hLTBP-3 (Ab-hL3/2925), (B) anti-ß1•LAP antibody (680) or (C) anti-LTBP-1 (ab39) antibodies. The signals for A and B are from the same filter, which was recycled. Black arrows indicate the large latent complexes of hLTBP-3 and ß1•LAP, whereas white arrows indicate the large latent complexes of LTBP-1 and ß1•LAP.

 


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  		Fig. 6. Proteolytic processing of hLTBP-3. Conditioned medium of COS-7 cells overexpressing the large latent complex of hLTBP-3 and TGF-ß1•LAP was treated with the indicated concentrations of plasmin and leukocyte elastase. The samples were analyzed by 7.5% SDS-PAGE and immunoblotting using Ab-hL3/2925 under non-reducing conditions.

 

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