Identification and characterisation of HDGF splice variants and their differential secretion via exosomal microvesicles

Abstract

Hepatoma-derived growth factor (HDGF) is a ubiquitously expressed protein and involved in a variety of cellular processes. Since HDGF exhibits growth stimulating activity, research focused mainly on its role in cancer and tumour biology. Interestingly, screening the NCBI database for HDGF entries led to the discovery of two further alternative human HDGF isoforms, termed HDGF-B and HDGF-C in the present work. Even though these entries have been present in the NCBI database for quite a while, the respective HDGF isoforms have been neglected in HDGF research by now. Therefore, main aim of this thesis was the verification and initial characterisation of those two novel HDGF isoforms. HDGF-B and HDGF-C arise from the usage of an alternative first in-frame exon and the expressed proteins exhibit changes in their N-terminal part. The remaining C-terminal part is identical to that of the basic HDGF form, now termed HDGF-A. Since most of the known interactions and functions of HDGF-A have been assigned to its N-terminal part, changes in this part of the protein might have a huge impact. Indeed, the results of this thesis indicated that the novel isoforms carry out quite distinct functions. This became particularly obvious regarding the analysis of co-precipitated proteins of each isoform. Only HDGF-A co-precipitated the RNA binding proteins nucleolin and YB-1, whereas HDGF-B and HDGF-C lost these interaction partners. Instead, these isoforms were able to co-precipitate tubulin and the motorprotein dynein and conversely these interaction partners were not detectable in the co-precipitate of HDGF-A. A further alteration was discovered in the secretion behaviour of these isoforms. HDGF-A is considered as a protein which leaves the cell via an alternative secretion pathway. Recently, some major proteomic studies detected its presence in exosomes, small secreted vesicles. This discovery was supported by the results of this thesis. Moreover it was found that HDGF-A is located within in the lumen of those extracellular vesicles. Interestingly, HDGF-A was additionally detected as free protein in the surrounding media, suggesting that it uses an additional alternative pathway to leave the cell. By contrast to this the novel isoforms HDGF-B and HDGF-C were detected at the surface of extracellular vesicles without any evidence that these proteins are additionally present as free protein. A remarkable finding of this thesis was the discovery that truncation of the outermost 15 N-terminal HDGF-A is sufficient to induce the same secretion and interaction behaviour as observed for HDGF-B and HDGF-C. The results highlighted the importance of the outermost N-terminal peptide in HDGF-A. A previous study revealed that N-terminal truncation might be controlled by dephosphorylation of serine residue 165 in HDGF-A. Assumed that such a truncation mechanism exists also under physiological conditions the cell is able to control the mode of secretion as well as interaction behaviour not only by alternative splicing but also by posttranslational modifications. This work provides insight into new aspects of HDGF, which help to understand its biological function and open up new perspectives for future studies.